LTBP COMPLEX-SPECIFIC INHIBITORS OF TGFb AND USES THEREOF

ABSTRACT

Disclosed herein are inhibitors, such as antibodies, and antigen-binding portions thereof, that selectively bind complexes of LTBP1-TGFβ and/or LTBP3-TGFβ. The application also provides methods of use of these inhibitors for, for example, inhibiting TGFβ activation, and treating subjects suffering from TGFβ-related disorders, such as fibrotic conditions. Methods of selecting a context-dependent or context-independent isoform-specific TGFβ inhibitor for a subject in need thereof are also provided.

RELATED APPLICATIONS

This international application claims the benefit of and priority toU.S. Provisional Application No. 62/798,927, filed Jan. 30, 2019, thecontents of which are expressly incorporated herein by reference inentirety.

BACKGROUND

Transforming growth factor beta (TGFβ) superfamily of growth factors areinvolved in a number of signaling cascades that regulate diversebiological processes including, but not limited to: inhibition of cellgrowth, tissue homeostasis, extracellular matrix (ECM) remodeling,endothelial to mesenchymal transition, cell migration and invasion, andimmune modulation/suppression, as well as mesenchymal to epithelialtransition. In relation to ECM remodeling, TGFβ signaling may increasefibroblast populations and ECM deposition (e.g., collagen). In theimmune system, TGFβ ligand modulates T regulatory cell function andmaintenance of immune precursor cell growth and homeostasis. In normalepithelial cells, TGFβ is a potent growth inhibitor and promoter ofcellular differentiation. However, as tumors develop and progress, theyfrequently lose their negative growth response to TGFβ. In this setting,TGFβ may become a promoter of tumor development due to its ability tostimulate angiogenesis, alter the stromal environment, and induce localand systemic immunosuppression. For these and other reasons, TGFβ hasbeen a therapeutic target for a number of clinical indications. Despitemuch effort made to date by a number of groups, clinical development ofa TGFβ therapeutic has been challenging.

Observations from preclinical studies, including in rats and dogs, haverevealed certain toxicities associated with inhibition of TGFβ in vivo.Moreover, although several TGFβ inhibitors have been developed to date,most clinical programs targeting TGFβ have been discontinued due to sideeffects or risk of toxicity.

For example, Anderton et al. (Toxicology Pathology, 39: 916-24, 2011)reported that small molecule inhibitors of TGFβ type I (ALK5) receptorinduced heart valve lesions characterized by hemorrhage, inflammation,degeneration and proliferation of valvular interstitial cells in apreclinical animal model. The toxicity was observed in all heart valvesat all doses tested. Frazier et al. (Toxicology Pathology, 35: 284-295,2007) reported that administration of the small molecule inhibitor ofTGFβ type I (ALK5) receptor GW788388 induced physeal dysplasia in rats.

Stauber et al. (J. Clin. Practice 4:3, 2014) reported that a chronic (≥3months) administration of the inhibitor of TGFβ receptor I kinase,LY2157299, which is being investigated for certain cancer treatments,caused multiple organ toxicities involving the cardiovascular,gastrointestinal, immune, bone/cartilage, reproductive, and renalsystems, in rats and dogs.

Fresolimumab (GC1008), a “pan” TGFβ antibody capable of neutralizing allhuman isoforms of TGFβ, has been reported to induce an epithelialhyperplasia of the gingiva, bladder, and of the nasal turbinateepithelium after multiple administrations in studies with cynomolgusmacaques (Lonning et al., Current Pharmaceutical Biotechnology 12:2176-89, 2011). Similarly, a variety of skin rashes/lesions, gingivalbleeding and fatigue have been reported in clinical trials afteradministration of multiple doses of the drug. The most notable adversereaction to fresolimumab includes the induction of cutaneouskeratoacanthomas and/or squamous cell carcinomas in human cancerpatients (see, for example: Lacouture et al., 2015, Cancer ImmunolImmunother, 64: 437-46; Stevenson et al., 2013, Oncolmmunology, 2:8,e26218; and Lonning et al., 2011). Additional evidence from a clinicaltrial suggests that in some cases this antibody may accelerate tumorprogression (Stevenson et al., 2013, Oncolmmunology, 2:8, e26218).

Thus, new methods and compositions for modulating TGFβ signaling arenecessary that can be used to effectively and safely treat diseases anddisorders involving TGFβ, including, for example, cancer, fibrosis andinflammation.

With an increasing recognition of potentially dangerous adverse effectsassociated with broad inhibition of TGFβ, a number of groups have morerecently turned to identifying inhibitors that target a subset—but notall—of the isoforms and still retain sufficient efficacy. For example,WO 2016/161410 discloses neutralizing antibodies that bind both TGFβ1and TGFβ2 (i.e., TGFβ1/2 inhibitors). WO 2006/116002 providesneutralizing antibodies that bind both TGFβ1 and TGFβ3 (i.e., TGFβ1/3inhibitors), albeit preferentially to the former. In addition totraditional monoclonal antibodies, some groups are developing engineeredfusion proteins that function as so-called “ligand traps” (see, forexample, WO 2018/158727, WO 2018029367 and WO 2018129331), at least someof which may be selective for TGFβ1/3. Another class of TGFβ1/3inhibitors include inhibitors of alpha-V (αv) integrins such asantibodies against αvβ6, which is an integrin known to activate bothTGFβ1 and TGFβ3 (i.e., TGFβ1/3). Yet others continue to pursue “better”pan-inhibitors that inhibit all three isoforms (i.e., TGFβ1/2/3 orpan-inhibitors) (see, for example, WO 2018/134681).

From an efficacy standpoint, however, the prevailing view of the fieldremains to be that it is advantageous to inhibit multiple isoforms ofTGFβ to achieve therapeutic effects, and to accommodate this, toxicitymanagement by “careful dosing regimen” is suggested as a solution(Brennan et al. (2018) mAbs, 10:1, 1-17).

Recently, Applicant described isoform-selective TGFβ1 inhibitors whichwere demonstrated to be both safe and efficacious in animal models (see,for example: WO 2017/156500 and WO 2018/129329, incorporated byreference), supporting the notion that selectively targeting the TGFβ1isoform, as opposed to broadly antagonizing all TGFβ isoforms, mayprovide an advantageous approach to achieving efficacy with acceptabletoxicity.

Whilst the observed safety profile achieved by selective inhibition ofTGFβ1 at doses that were shown efficacious in vivo is a promising steptowards developing a TGFβ1 inhibitor for clinical applications,identification of TGFβ1 inhibitors that are capable of selectivelyaffecting a defined subset of TGFβ1 effects (e.g., TGFβ inhibitors thatare selective to LTBP-presented complexes) remained elusive. Morerecently, Applicant demonstrated that such “LTBP context-specific”inhibitors can be generated (WO 2019/023661, incorporated herein byreference) using the methods previously described by Applicant (see, forexample, WO 2014/074532 and WO 2014/182676). However, the LTBP-selectiveTGFβ1 inhibitors described in the aforementioned internationalpublication showed modest affinities and inhibitory activities, coupledwith suboptimal cross-species reactivity.

SUMMARY OF THE INVENTION

The present disclosure provides improved TGFβ inhibitors capable ofselectively targeting matrix-associated proTGFβ complexes, such asLTBP1-proTGFβ1 and LTBP3-proTGFβ1.

These inhibitors bind and inhibit LTBP1- and/or LTBP3-presentedproTGFβat high affinities (at least nanomolar range) but do not bind andinhibit immune cell-associated TGFβ, e.g., GARP-and/or LRRC33-presentedproTGFβ1, or the binding is below meaningful levels (e.g., at least 50times affinities for the LTBP complexes over GARP or LRRC33 complex).Thus, these inhibitors can selectively inhibit activation of TGFβ in acontext-dependent manner, such that they selectively bind, therebyinhibiting the TGFβ signaling axis associated with the ECM. Inparticular, the present disclosure includes selective inhibitors ofmatrix-associated (e.g., LTBP1 and/or LTBP3-associated) TGFβ activation.In some embodiments, such inhibitors specifically bind a particularisoform of TGFβ (e.g., proTGFβ1, proTGFβ2, and/or proTGFβ3) associatedwith LTBP1 and/or LTBP3, thus also providing TGFβ isoform specificity.In a particular embodiment, such inhibitors specifically bind toLTBP1/3-proTGFβ1. In any of the embodiments of the present invention,such inhibitors do not inhibit activation of TGFβ1 associated withimmune cell function, mediated by GARP and/or LRRC33. The improvedantibodies encompassed by the present disclosure have affinities towardshuman LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 in at least a nanomolarrange (i.e., 1×10 ⁹M to 10×10⁻⁹M). In some embodiments, such antibodiesalso have affinities towards murine LTBP1-proTGFβ1 and/or murineLTBP3-proTGFβ1 in at least a nanomolar range (i.e., 1×10⁻⁹M to10×10⁻⁹M).

Rationale for the therapeutic use of a TGFβ1 inhibitor that does nottarget the GARP-proTGFβ1 complex on regulatory T cells is at leastthreefold:

First, regulatory T cells play a crucial role in maintaining immunetolerance to self-antigens and in preventing autoimmune disease. SinceTregs generally suppress, dampen or downregulate induction andproliferation of effector T cells, systemic inhibition of this functionmay lead to overactive or exaggerated immune responses in the host bydisabling the “break” that is normally provided by Treg cells. Thus, theapproach taken here (e.g., TGFβ1 inhibition without disabling Tregfunction) is aimed to avoid the risk of eliciting autoimmunity.Furthermore, patients who already have a propensity for developingover-sensitive immune responses or autoimmunity may be particularly atrisk of triggering or exacerbating such conditions, without theavailability of normal Treg function; and therefore, the inhibitors thatselectively target the matrix TGFβ1 may advantageously minimize suchrisk.

Second, evidence suggests that an alteration in the Th17/Treg ratioleads to an imbalance in pro-fibrotic Th17 cytokines, which correlatewith severity of fibrosis, such as liver fibrosis (see, for example,Shoukry et al. (2017) J Immunol 198 (1 Supplement): 197.12). The presentinventors reasoned that perturbation of the GARP arm of TGFβ1 functionmay directly or indirectly exacerbate fibrotic conditions.

Third, regulatory T cells are indispensable for immune homeostasis andthe prevention of autoimmunity. It was reasoned that, particularly for aTGFβ1 inhibition therapy intended for a long-term or chronicadministration, it would be desirable to avoid potential side effectsstemming from perturbation of normal Treg function in maintaining immunehomeostasis (reviewed in, for example, Richert-Spuhler and Lund (2015)Prog Mol Biol Transl Sci. 136: 217-243). This strategy is at least inpart aimed to preserve normal immune function, which is required, interalia, for combatting infections.

To this end, the inventors of the present disclosure set out to generateisoform-specific, context-selective inhibitors of TGFβ1 that selectivelytarget matrix-associated TGFβ1 activation but not immune cell-associatedTGFβ1 activation.

Technical challenges that exist to date include limited ability todiscern and selectively modulate these subpools of TGFβ1 present invarious contexts (or “niches”) in vivo.

In an effort to address this challenge, the present inventors haveidentified isoform-specific monoclonal antibodies that bind the latentTGFβ1 prodomain, with no detectable binding to latent TGFβ2 or TGFβ3,and that inhibit integrin-mediated activation of latent TGFβ1 in vitrowith the context-dependency as described herein. The discovery andcharacterization of such antibodies was made possible, at least in part,by the development of context-dependent cell-based assays of TGFβ1activation. In the process of this novel assay development andvalidation, it was demonstrated that, like the αVβ6 integrin, αVβ8 canalso activate LTBP1-proTGFβ1. It was further demonstrated that, similarto the LTBP1 complex, LTBP3-proTGFβ1 can be activated by αVβ6.Antibodies discovered by screening in these assays revealed a class ofantibodies that binds and inhibits TGFβ1 only when presented by LTBP1 orLTBP3. Such LTBP-specific antibodies do not inhibit TGFβ1 in the contextof the immune-associated TGFβ1 presenters GARP and LRRC33. Suchantibodies are therapeutic candidates for the treatment of disordersincluding, e.g., fibrotic conditions, and could allow chronic dosingthat would avoid TGFβ-related immune system activation. Methods ofselecting a context-specific or context-independent TGFβ1 inhibitor forvarious fibrotic conditions are also provided herein.

Accordingly, in one aspect, the invention provides isoform-specific TGFβantibodies, or antigen-binding fragments thereof, characterized in thatthey bind selectively to an LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1complex with a K_(D)≤50 nM. In one embodiment, the invention providesisoform-specific TGFβ antibodies, or antigen-binding fragments thereof,characterized in that they bind selectively to an LTBP1-TGFβ1 complexand/or a LTBP3-TGFβ1 complex with a K_(D)≤25 nM. In one embodiment, theinvention provides isoform-specific TGFβ antibodies, or antigen-bindingfragments thereof, characterized in that they bind selectively to anLTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with a K_(D)≤10 nM. Inone embodiment, the invention provides an isolated antibody, orantigen-binding portion thereof, that selectively binds to aLTBP1-proTGFβ1 complex and a LTBP3-proTGFβ1 complex, wherein theantibody, or antigen-binding portion thereof, does not bind to one ormore of the following targets: (a) LTBP1 alone; (b) proTGFβ1 alone; (c)a GARP-proTGFβ1 complex; and (d) a LRRC33-proTGFβ1 complex. In furtherembodiments, the invention provides isoform-specific TGFβ antibodies, orantigen-binding fragments thereof, characterized in that they bindselectively to an LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex witha K_(D)≤5 nM.

In one aspect, the invention provides inhibitors of extracellularmatrix-associated TGFβ activation, which selectively bind aLTBP1/3-presented proTGFβ latent complex. In one embodiment, theinhibitor does not inhibit immune cell-associated TGFβ1 activation, forexample, immune cell-associated TGFβ1 activation that results fromactivation of a GARP-presented proTGFβ1 latent complex. In exemplaryembodiments, the inhibitor is an antibody, or antigen-binding portionthereof.

In other aspects, the invention provides TGFβ antibodies, orantigen-binding fragments thereof, characterized in that they bindselectively to an LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex. Insome embodiments, the antibodies, or antigen-binding fragments thereof,selectively bind to LTBP1-TGFβ1. In some embodiments, such antibodiesbind both human and murine counterparts.

In one aspect, the invention provides an isolated antibody, orantigen-binding portion thereof, that selectively binds an LTBP1-proTGFβlatent complex and/or an LTBP3-proTGFβ latent complex, therebymodulating release of mature TGFβ growth factor from the latent complex,wherein the antibody, or antigen-binding portion thereof, does not bindmature TGFβ1 alone or a GARP-proTGFβ1 latent complex. In one embodiment,the antibody, or antigen-binding portion thereof, does not bind anLRRC33-proTGFβ1 latent complex. Alternatively, in one embodiment, theantibody, or antigen-binding portion thereof, binds an LRRC33-proTGFβ1latent complex.

In some embodiments, the antibody, or antigen-binding portion thereof,is specific to an LTBP1-proTGFβ1 latent complex. In other embodiments,the antibody, or antigen-binding portion thereof, is specific to anLTBP3-proTGFβ1 latent complex. In one embodiment, the antibody, orantigen-binding portion thereof, binds an LTBP1-proTGFβ1 complex and/ora LTBP3-proTGFβ1 complex with a dissociation constant (K_(D)) of atleast about 10⁻⁸ M. In one embodiment, the antibody, or antigen-bindingportion thereof, binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex with a K_(D) of <50 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI). In oneembodiment, the antibody, or antigen-binding portion thereof, binds ahuman LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex witha K_(D) of <10 nM as measured in a suitable in vitro binding assay suchas Bio-Layer Interferometry (BLI). In one embodiment, the antibody, orantigen-binding portion thereof, binds a mouse LTBP1-proTGFβ1 complexand/or a mouse LTBP3-proTGFβ1 complex with a K_(D) of <50 nM as measuredin a suitable in vitro binding assay such as Bio-Layer Interferometry(BLI). In one embodiment, the antibody, or antigen-binding portionthereof, binds a mouse LTBP1-proTGFβ1 complex and/or a mouseLTBP3-proTGFβ1 complex with a K_(D) of <10 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI).

The present disclosure further provides antibodies and antigen bindingfragments thereof, which selectively bind an LTBP1-proTGFβ complexand/or an LTBP3-proTGFβ complex and have one or more yet furtheradvantageous properties. Indeed, the inventors surprisingly found thatsuch antibodies could be provided which bind a human LTBP1-proTGFβcomplex and a human LTBP3-proTGFβ complex with high affinity, andadvantageously slow dissociation rates, while also being cross-reactivewith mouse LTBP1-proTGFβ complex and mouse LTBP3-proTGFβ complex anddisplaying no significant binding to human GARP-proTGFβ complex (orindeed to human LRRC33-proTGFβ complex).

Further still, antibodies disclosed herein (including antibodies havingone or more, or even all of the aforementioned advantageous properties)exhibit potent inhibition of TGFβ1 signaling in cell-based assays, andsignificantly reduce markers of fibrosis and TGFβ signaling in multipleanimal models of fibrosis.

Thus, in some embodiments, the antibody, or antigen-binding fragmentthereof binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1complex with a K_(D) of <5 nM as measured by BLI, and has one or more ofthe following properties:

-   -   i) is cross-reactive with mouse LTBP1-proTGFβ1 complex;    -   ii) is cross-reactive with mouse LTBP3-proTGFβ1 complex;    -   iii) binds a mouse LTBP1-proTGFβ1 complex with a K_(D) of <10 nM        as measured by BLI;    -   iv) binds a mouse LTBP3-proTGFβ1 complex with a K_(D) of <10 nM        as measured by BLI;    -   v) binds a human LTBP1-proTGFβ1 complex and/or a human        LTBP3-TGFβ1 complex with a K_(D) that is at least 50 times lower        than the K_(D) when binding to a human GARP-proTGFβ1 complex        under the same assay conditions;    -   vi) does not show detectable binding to a human GARP-proTGFβ1        complex, as measured by BLI, under the same assay conditions as        used to measure binding to human LTBP1-proTGFβ1 complex and/or a        human LTBP3-TGFβ1 complex;    -   vii) does not show detectable binding to an LRRC33-proTGFβ1        complex (e.g., a human

LRRC33-proTGFβ1 complex) as measured by BLI, under the same assayconditions as used to measure binding to human LTBP1-proTGFβ1 complexand/or human LTBP3-TGFβ1 complex.

In some embodiments, the antibody or antigen-binding fragment has atleast properties (i)-(v) above, and optionally (vii). In someembodiments, the antibody or antigen-binding fragment has at leastproperties (i)-(iv) and (vi) above, and optionally (vii). In someembodiments, the antibody or antigen-binding fragment has at leastproperties (i), (iii) and (v) above, and optionally (vii). In someembodiments, the antibody or antigen-binding fragment has at leastproperties (ii), (iv) and (v) above, and optionally (vii). In someembodiments, the antibody or antigen-binding fragment has at leastproperties (i), (iii) and (vi) above, and optionally (vii). In someembodiments, the antibody or antigen-binding fragment has at leastproperties (ii), (iv) and (vi) above, and optionally (vii). In someembodiments, the antibody or antigen-binding fragment has at leastproperties (i)-(iii) and (v) above, and optionally (vii). In someembodiments, the antibody or antigen-binding fragment has at leastproperties (i)-(iii) and (vi) above, and optionally (vii).

In some preferred embodiments, the antibody or antigen-binding fragmentbinds a human LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complexwith a K_(D) of <5 nM as measured by BLI, and has all of the aboveproperties (i)-(vii).

The antibody or antigen-binding fragment may selectively bind aLTBP1/3-presented proTGFβ latent complex and inhibit extracellularmatrix-associated TGFβ activation.

Further still, further advantageous isoform-selective inhibitors ofTGFβ1 activation may include monoclonal antibodies (includingimmunoglobulins and antigen-binding fragments or portions thereof) thatexhibit slow dissociation rates (i.e., off-rates, k_(OFF)). Thus, theinvention is further based on the recognition that treatment of chronicand progressive disease such as fibrosis may require inhibitors withsuperior durability, which may be reflected on the dissociation rate ofsuch antibody.

The affinity of an antibody to its antigen is typically measured as theequilibrium dissociation constant, or K_(D). The ratio of theexperimentally measured off- and on-rates (k_(OFF)/k_(ON)) can be usedto calculate the K_(D) value. The k_(OFF) value represents the antibodydissociation rate, which indicates how quickly it dissociates from itsantigen, whilst the k_(ON) value represents the antibody associationrate which provides how quickly it binds to its antigen. The latter istypically concentration-dependent, while the former isconcentration-independent. The K_(D) value relates to the concentrationof antibody (the amount of antibody needed for a particular experiment)and so the lower the K_(D) value (lower concentration) and thus thehigher the affinity of the antibody. With respect to a referenceantibody, a higher affinity antibody may have a lower k_(OFF) rate, ahigher k_(ON) rate, or both.

Both the k_(OFF) and k_(ON) rates contribute to the overall affinity ofa particular antibody to its antigen, and relative importance or impactof each component may depend on the mechanism of action of the antibody.For example, neutralizing antibodies, which bind mature growth factors(e.g., soluble, transient TGFβ1 ligand liberated from a latent complex),must compete with the endogenous high-affinity receptors for ligandbinding in vivo. Because the ligand-receptor interaction is a localevent and because the ligand is short-lived, such antibodies must becapable of rapidly targeting and sequestering the soluble growth factorbefore the ligand finds its cellular receptor—thereby activating theTGFβ1 signaling pathway—in the tissue. Therefore, for ligand-targetingneutralizing antibodies to be potent, the ability to bind the targetgrowth factor fast, i.e., high association rates (k_(ON)), may beespecially important.

By contrast, Applicant reasoned that antibodies that inhibit the TGFβ1signaling by preventing the activation (e.g., release) of mature growthfactor from the latent complex (“activation inhibitors”) maypreferentially benefit from having slow dissociation rates once theantibody is engaged with the target antigen (e.g., proTGFβ1 complexes).Unlike neutralizing antibodies, such antibodies do not directly competewith cellular receptors; rather, they work upstream of the signaling bytargeting inactive precursor forms (e.g., latent proTGFβ1 complexes)that remain dormant within a tissue environment thereby preemptivelypreventing the activation of TGFβ1. Such antibodies may exert theirinhibitory activity by preventing mature growth factor from beingliberated from the latent complex. For example, such antibodies mayfunction like a “clamp” to lock the active growth factor in theprodomain cage structure to keep it in an inactive (e.g., “latent”)state. Indeed, structural analyses, including epitope mapping, providedinsight into the molecular mechanism underlining the ability of theseantibodies to block TGFβ1 activation. In this regard, the Latency Lassoregion of the prodomain may be a particularly useful target.

Upon target engagement, antibodies that are able to remain bound to thetarget (e.g., dissociate very slowly from the latent complex) areexpected to be advantageous in achieving superior in vivo potency, dueto enhanced durability of effects and/or avidity. Based on thisrecognition,

Applicant of the present disclosure sought to identify isoform-selectiveactivation inhibitors of TGFβ1 with particularly low k_(OFF) values ascompared to previously described antibodies. Thus, according to theinvention, preferred antibodies have high affinities primarilyattributable to a slow dissociation rate (k_(OFF)), as opposed to fastassociation rate (k_(ON)). Accordingly, in some embodiments, theantibody, or antigen-binding fragment thereof binds a humanLTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K_(D)of <5 nM as measured by BLI, and has one or more of the followingproperties (which may be in addition to one of properties (i)-(vii), orcombinations thereof set out above):

(viii) low dissociation rates (k_(OFF)) of <5×10⁻⁴ (1/s), when bindinghuman LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1 complex (e.g., asmeasured by a suitable in vitro binding/kinetics assay, such as by BLI,e.g., Octet-based systems); and/or

(ix) long half-binding time (t½) of ≥45 minutes when bound to humanLTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 complex (e.g., as measured bySPR).

In some preferred embodiments, the antibody or antigen binding fragmentcomprises the following six CDRs:

-   -   a) CDR-H1 comprising the amino acid sequence FTFRSYVMH (SEQ ID        NO: 166);    -   b) CDR-H2 comprising the amino acid sequence        VISHEGS(X₁)KYYADSVKG, wherein: X₁ is L or G (SEQ ID NO: 366);        and    -   c) CDR-H3 comprising the amino acid sequence        A(X₁)PRIAARRGGFG(X₂), wherein: X₁ is V, R or L; and X₂ is Y, S        or T (SEQ ID NO: 367);    -   d) CDR-L1 comprising the amino acid sequence        TRS(X₁)G(X₂)ID(X₃)NYVQ, wherein, X₁ is S or H; X₂ is N, L, S or        A; and X₃ is N, D or Y (SEQ ID NO: 368);    -   e) CDR-L2 comprising the amino acid sequence ED(X₁)(X₂)RPS,        wherein: X₁ is N, F or A; and X₂ is Q, I or V (SEQ ID NO: 369);        and    -   f) CDR-L3 comprising the amino acid sequence        Q(X₁)YD(X₂)(X₃)(X₄)Q(X₅)VV, wherein: X₁ is S or G; X₂ is S, F,        Y, D, H or W; X₃ is N, D or S; X₄ is N, A, L, E or T; and X₅ is        G, R, A or L (SEQ ID NO: 370).

In some preferred embodiments, the antibody or antigen-binding fragmentthereof competes or cross-competes with an antibody having a heavy chainvariable region sequence as set forth in SEQ ID NO: 318 and light chainvariable region sequence as set forth in SEQ ID NO: 319 (e.g., Ab42).The antibody may comprise a heavy chain variable region having an aminoacid sequence that is at least 90% identical to SEQ ID NO: 318 and alight chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 319.

The antibody or antigen-binding fragment thereof provided herein may, insome preferred embodiments, comprise the following six CDRs (e.g., thoseof Ab42):

CDR-H1 comprising the amino acid sequence FTFRSYVMH (SEQ ID NO: 166);

CDR-H2 comprising the amino acid sequence VISHEGSLKYYADSVKG (SEQ ID NO:167);

CDR-H3 comprising the amino acid sequence ARPRIAARRGGFGY (SEQ ID NO:168);

CDR-L1 comprising the amino acid sequence TRSSGNIDNNYVQ (SEQ ID NO:169);

CDR-L2 comprising the amino acid sequence EDNQRPS (SEQ ID NO: 170); and

CDR-L3 comprising the amino acid sequence QSYDYDTQGVV (SEQ ID NO: 171).

The antibody or antigen-binding fragment may further comprise a heavychain variable region having an amino acid sequence that is at least 95%identical (optionally at least 98% identical) to SEQ ID NO: 318 and alight chain variable region having an amino acid sequence that is atleast 95% identical (optionally at least 98% identical) to SEQ ID NO:319.

In some alternative embodiments, the antibody, or antigen-bindingfragment thereof, comprises the following six CDRs:

1a) CDR-H1 comprising the amino acid sequence GSIRSSSYYWG (SEQ ID NO:292);

-   -   b) CDR-H2 comprising the amino acid sequence SISYSATTYY (SEQ ID        NO: 293);    -   c) CDR-H3 comprising the amino acid sequence        A(X₁)DPSYDS(X₂)AGM(X₃)V, wherein: X₁ is S or G; X₂ is A or I;        and X₃ is D or Q (SEQ ID NO: 371);    -   d) CDR-L1 comprising the amino acid sequence        RAS(X₁)(X₂)IS(X₃)YLN, wherein: X₁ is K or Q; X₂ is V or S; and        X₃ is S or Y (SEQ ID NO: 389);    -   e) CDR-L2 comprising the amino acid sequence (X₁)AS(X₂)(X₃)QS,        wherein: X₁ is Y,

A or S; X₂ is S or N; and X₃ is L or R (SEQ ID NO: 390);

-   -   f) CDR-L3 comprising the amino acid sequence        QQ(X₁)(X₂)D(X₃)P(X₄)T, wherein: X₁ is S or G; X₂ is F or N; X₃        is W or F; and X₄ is F or L (SEQ ID NO: 391).

In some embodiments, the antibody or antigen-binding fragment thereofcompetes or cross-competes with an antibody having a heavy chainvariable region sequence as set forth in SEQ ID NO: 360 and light chainvariable region sequence as set forth in SEQ ID NO: 361 (e.g., Ab63).The antibody may comprise a heavy chain variable region having an aminoacid sequence that is at least 90% identical to SEQ ID NO: 360 and alight chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 361.

The antibody or antigen-binding fragment thereof provided herein maycomprise the following six CDRs (e.g., those of Ab63):

CDR-H1 comprising the amino acid sequence GSIRSSSYYWG (SEQ ID NO: 292);

CDR-H2 comprising the amino acid sequence SISYSATTYY (SEQ ID NO: 293);

CDR-H3 comprising the amino acid sequence AGDPSYDSIAGMQV (SEQ ID NO:294);

CDR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO: 295);

CDR-L2 comprising the amino acid sequence AASNLQS (SEQ ID NO: 296); and

CDR-L3 comprising the amino acid sequence QQSFDWPLT (SEQ ID NO: 297).

The antibody or antigen-binding fragment may further comprise a heavychain variable region having an amino acid sequence that is at least 95%identical (optionally at least 98% identical) to SEQ ID NO: 360 and alight chain variable region having an amino acid sequence that is atleast 95% identical (optionally at least 98% identical) to SEQ ID NO:361.

In one aspect, the invention provides an antibody, or antigen-bindingfragment thereof, for use in a method for treating a fibrotic disorderin a subject, wherein the antibody, or antigen-binding fragment thereof,specifically binds a human LTBP1-proTGFβ complex and/or a humanLTBP3-proTGFβ complex, and does not bind a human GARP-proTGFβ1 complex;, wherein: a) the fibrotic disorder comprises chronic inflammation; b)the subject benefits from immune suppression; c) the subject has or isat risk of developing an autoimmune disease; d) the subject is acandidate for or has received an allograft transplant; e) the subjecthas an elevated Th17/Treg ratio; and/or, f) the subject is in need of along-term or chronic administration of the TGFβ1 inhibitor. In someembodiments, the the subject has or is at risk of developing a metabolicdisorder (and the subject is optionally a subject according to one ormore of a)-f)). In some embodiments, the the antibody, orantigen-binding fragment thereof, is an isoform-specific LTBP1-proTGFβ1inhibitor and/or LTBP3-proTGFβ1 inhibitor.

The antibodies or antigen-binding fragments therof provided herein maybe used in a method for treating a fibrotic disorder in a subject. Thefibrotic disorder may comprise chronic inflammation. The subject maybenefit from immune suppression. The subject may have or be at risk ofdeveloping an autoimmune disease. The subject may be a candidate for ormay have received an allograft transplant.

Alternatively, or in addition, the subject may have an elevatedTh17/Treg ratio. The subject may be in need of a long-term or chronicadministration of the TGFβ1 inhibitor.

Alternatively, or in addition, the subject may have or be at risk ofdeveloping a metabolic disorder.

In another aspect, the invention provides a method for making acomposition comprising an antibody, or antigen-binding fragment thereof,that specifically binds a human LTBP1-proTGFβ complex and/or a humanLTBP3-proTGFβ complex, and does not bind a human GARP-proTGFβ1 complex;wherein the antibody, or antigen-binding fragment thereof, inhibitsTGFβ1 but does not inhibit TGFβ2 or TGFβ3, the method comprising stepsof i) providing at least one antigen comprising LTBP1-proTGFβ1 and/orLTBP3-proTGFβ1, ii) selecting a first pool of antibodies, orantigen-binding fragments thereof, that specifically bind the at leastone antigen of step (i) so as to provide specific binders ofLTBP1-proTGFβ1 and/or LTBP3-proTGFβ1; iii) selecting a second pool ofantibodies, or antigen-binding fragments thereof, that inhibitactivation of TGFβ1, so as to generate specific inhibitors of TGFβ1activation; iv) formulating an antibody, or antigen-binding fragmentthereof, that is present in the first pool of antibodies and the secondpool of antibodies into a pharmaceutical composition, thereby making thecomposition comprising the antibody, or antigen-binding fragmentthereof.

In one embodiment, the method further comprises a step of removing fromthe first pool of antibodies, or antigen-binding fragments thereof, anyantibodies, or antigen-binding fragments thereof, that bindGARP-proTGFβ1, LRRC33-proTGFβ1, mature TGFβ1, GARP-proTGFβ2,LRRC33-proTGFβ2, mature TGFβ2, GARP-proTGFβ3, LRRC33-proTGFβ3, matureTGFβ3, or any combinations thereof. In one embodiment, the methodfurther comprises a step of determining or confirmingisoform-specificity of the antibodies, or antigen-binding fragmentsthereof, selected in steps (ii) and/or (iii). In one embodiment, themethod further comprises a step of selecting for antibodies, orantigen-binding fragments thereof, that are cross-reactive to human androdent antigens. In one embodiment, the method further comprises a stepof generating a fully human or humanized antibody, or antigen-bindingfragment thereof, of the antibody, or antigen-binding fragment thereof,that is present in the first pool of antibodies and the second pool ofantibodies.

In one embodiment, the method further comprises a step of subjecting theantibody, or antigen-binding fragment thereof, that is present in thefirst pool of antibodies and the second pool of antibodies to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody or fragment thereof. In one embodiment, theaffinity maturation/optimization comprises a step of subjecting theantibody, or antigen-binding fragment thereof, that is present in thefirst pool of antibodies and/or the second pool of antibodies to lightchain shuffling as described herein. In one embodiment, the affinitymaturation/optimization comprises the step of subjecting the antibody,or antigen-binding fragment thereof, that is present in the first,second, and/or third pool of antibodies to CDR H1/H2 diversification asdescribed herein. In one embodiment, the affinitymaturation/optimization comprises the step of subjecting the antibody,or antigen-binding fragment thereof, to CDR-H3 mutagenesis as describedherein. In one embodiment, the affinity maturation/optimizationcomprises the step of subjecting the antibodies, or antigen-bindingfragment thereof, to light chain CDR mutagenesis as described herein. Inone embodiment, the affinity maturation/optimization comprises the stepof subjecting the antibodies, or antigen-binding fragment thereof, tolight chain CDR L1/L2 diversification as described herein.

In one embodiment, the method further comprises a step of determiningaffinity of the antibodies, or antigen-binding fragments thereof, fromthe first and/or second pools of antibodies to human LTBP1-proTGFβ1and/or human LTBP3-proTGFβ1. In some embodiments, the method furthercomprises a step of removing from the first and/or second pools ofantibodies, or antigen-binding fragments thereof, any antibodies, orantigen-binding fragments thereof, that bind to human LTBP1-proTGFβ1and/or human LTBP3-proTGFβ1 with a K_(D) of >100 nM, >50 nM, >25 nM,or >10 nM, as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI).

In one embodiment, the method further comprises a step of determiningaffinity of the antibodies, or antigen-binding fragments thereof, fromthe first and/or second pools to mouse LTBP1-proTGFβ1 and/or mouseLTBP3-proTGFβ1. In some embodiments, the method further comprises a stepof removing from the first and/or second pools of antibodies, orantigen-binding fragments thereof, any antibodies, or antigen-bindingfragments thereof, that bind to mouse LTBP1-proTGFβ1 and/or mouseLTBP3-proTGFβ1 with a K_(D) of >100 nM, >50 nM, or >10 nM, as measuredin a suitable in vitro binding assay such as Bio-Layer Interferometry(BLI).

In one embodiment, the method further comprises a step of removing fromthe first and/or second pools of antibodies, or antigen-bindingfragments thereof, any antibodies, or antigen-binding fragments thereof,that do not bind mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1.

In one embodiment, the method further comprises a step of determiningthe IC₅₀ of the antibodies, or antigen-binding fragments thereof, fromof the first and/or second pools of antibodies, or antigen-bindingfragments thereof, as measured by a suitable functional in vitrocell-based assay such as a caga assay, as described herein. In someembodiments, the method comprises the step of removing antibodies, orantigen-binding fragments thereof, from the first and/or second pools ofantibodies, or antigen-binding fragments thereof, that have an IC₅₀ ofgreater than 100 nM, 50 nM, 25 nM, 10 nM, or 5 nM as measured by acell-based assay (such as a caga assay) as described herein.

In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof, from the first and/orsecond pools, antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 50 nM or 10 nM as measured by an endogenousLTBP caga assay as described herein.

In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof, from the first and/orsecond pools, antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 50 nM, 25 nM, or 10 nM, as measured by ahuman LTBP overexpression caga assay as described herein.

In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof, from the first and/orsecond pools, antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 50 nM, 25 nM, 10 nM, or 5 nM, as measuredby a murine LTBP overexpression caga assay as described herein.

Processes and methods for identifying or selecting TGFβ1-selectiveinhibitors suitable for therapeutic use are encompassed by theinvention, as are methods for making a composition comprising aTGFβ1-selective inhibitor. In preferred embodiments, a TGFβ1 inhibitor(e.g., a selected inhibitor) includes one or more antibodies orantigen-binding fragments with particularly advantageous kineticscriteria characterized by: i) high affinities to each of humanLTBP1/3-proTGFβ1 complexes (e.g., K_(D)≤5 nM), and, ii) low dissociationrates (k_(OFF)), e.g., ≤5×10⁻⁴ (1/s), as measured by a suitable in vitrobinding/kinetics assay, such as by BLI, e.g., Octet-based systems. Thelow dissociation rate criterion may be reflected in long dissociationhalf-time t½), e.g., >45 minutes from human LTBP1-proTGFβ1 and/or humanLTBP3-proTGFβ1 complexes. Preferably, the long dissociation half-time ofan antibody or antigen-binding fragment thereof for thematrix-associated complex(es) is coupled with short dissociationhalf-time with respect to cell-assosciated complexes, e.g., humanGARP-proTGFβ1 and/or human LRRC33-proTGFβ1 complexes. In particular,preferred antibodies or fragments dissociate from human GARP-proTGFβ1complex with t½ of no more than 10 minutes, more preferably no more than5 minutes. Likewise, methods for making a composition comprising aTGFβ1-selective inhibitor as described herein may further include a stepof selecting such antibodies. The selected antibody or the plurality ofantibodies are evaluated in preclinical studies comprising an efficacystudy and a toxicology/safety study, employing suitable preclinicalmodels. Effective amounts of the antibody or the antibodies determinedin the efficacy study are below the level that results in undesirabletoxicities determined in the toxicology/safety study. Preferably, theantibody or antibodies are selected which has/have at least 3-fold,6-fold, and more preferably 10-fold therapeutic window. Effectiveamounts of the antibodies according to the present disclosure may bebetween about 0.1 mg/kg and about 30 mg/kg when administered weekly. Inpreferred embodiments, the maximally tolerated dose (MTD) of theantibodies according to the present disclosure is >100 mg/kg when dosedweekly for at least 4 weeks. In some embodiments, in a preclinicaltoxicology study, the antibodies show a NOAEL of >100 mg/kg/week, >200mg/kg/week or >300 mg/kg/week, wherein optionally the toxicology studyis a 4-week study, 8-week study, or a 12-week study. For example, theNOAEL is >100 mg/kg/week in a 12-week sub-chronic dosing regimen inhealthy mice or rats.

The present disclosure also includes a surprising finding thatinhibition of TGFβ3 with a TGFβ3-selective inhibitor produced profibrotic effects in mice. Similarly, concurrent inhibition of both TGFβ1and TGFβ3 in the same model with a combination of a TGFβ1-selectiveinhibitor and a TGFβ3-selective inhibitor resulted in attenuatedanti-fibrotic effects of the TGFβ1 inhibitor. These observations raisethe possibility that non-selective TGFβ inhibitors (such aspan-inhibitors and TGFβ1/3 inhibitors) may in fact exacerbate fibrosis.Advantageously, the antibodies disclosed herein (e.g., Ab42 and variantsthereof, as described herein) are isoform-selective in that theyspecifically target the latent TGFβ1 complex and do so with lowdissociation rates. Thus, the invention includes the recognition thatwhen selecting a particular TGFβ inhibitor for patients with a fibroticcondition (e.g., disease involving ECM dysregulation), isoformselectivity should be carefully considered so as to avoid risk ofexacerbating ECM dysregulation. Accordingly, the present disclosureincludes therapeutic methods comprising selecting a TGFβ inhibitor thatdoes not inhibit TGFβ3 to treat a subject with a fibrotic condition,(including preferred fibrotic conditions, as described herein).

The isoform-selective LTBP1/3-proTGFβ1 complex-selective inhibitor asused herein may in some embodiments be selected from Ab31, Ab34, Ab37,Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab62, Ab63, and Ab64(optionally Ab42 or Ab63) (i.e., an antibody or antigen-binding fragmenthaving the heavy and light chain variable regions of the correspondingAb, as provided herein), a variant/derivative or antigen-bindingfragment thereof thereof, or an engineered molecule comprising anantigen-binding fragment thereof. In some preferred embodiments, theLTBP1/3-proTGFβ1 complex-selective inhibitor inhibitor is Ab42, avariant/derivative or antigen-binding fragment thereof, or an engineeredmolecule comprising an antigen-binding fragment thereof. In preferredembodiments, the LTBP1/3-proTGFβ1 complex-selective inhibitor is Ab42 oran antigen-binding fragment thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically depicts that targeting of the latent form of TGFβ1provides isoform and context specificity.

FIGS. 2A-2B demonstrate the identification of isoform-specific and LTBPcomplex-specific binders of latent TGFβ1. FIG. 2A demonstrates thatSR-AB1 binds latent TGFβ1, independent of the presenting molecule.SR-AB1 is a human monoclonal antibody that was discovered by yeastdisplay, which selectively binds latent TGFβ1, without detectablebinding to latent TGFβ2, TGFβ3, or mature TGFβ1. SR-AB1 cross-reactswith mouse, rat, and cynomolgus monkey proteins and binds to all fourlatent TGFβ1 complexes. FIG. 2B demonstrates that SR-AB2, ananti-LTBP1-proTGFβ1 antibody, does not bind GARP-proTGFβ1 or matureTGFβ1. SR-AB2 cross-reacts with rodent LTBP1-proTGFβ1.

FIGS. 3A-3B demonstrate functional assays (potency assays) to detect theinhibition of activated recombinant latent TGFβ1. FIG. 3A depicts theactivation of latent TGFβ1 deposited in the extracellular matrix (ECM).In this assay, presenting molecules are co-transfected with proTGFβ1 inintegrin-expressing cells. Transiently transfected cells are seeded inassay plates in the presence of inhibitors. Latent LTBP-proTGFβ1 complexis embedded in the ECM. TGFβ reporter cells are then added to thesystem; free growth factor (released by integrin) signals and isdetected by luciferase assay. FIG. 3B depicts the activation of latentTGFβ1 presented on the cell surface. Presenting molecules areco-transfected with proTGFβ1 in integrin-expressing cells. Latent TGFβ1is expressed on the cell surface by GARP or LRRC33. TGFβ reporter cellsand inhibitors are then added to the system; free growth factor(released by integrin) signals and is detected by luciferase assay.

FIGS. 4A-4B depict the optimization of the recombinant functionalassays. FIG. 4A depicts the relative contribution of presenting moleculeand/or proTGFβ1 activation upon co-transfection of presenting moleculeand proTGFβ1. FIG. 4B depicts the optimization of co-transfection: theratio of plasmid DNAs for presenting molecule and proTGFβ1. Equivalentamounts of each plasmid were optimal for co-transfection.

FIG. 5 demonstrates that fibronectin promotes integrin activation ofLTBP-presented latent TGFβ1. Assay plates were pre-coated withfibronectin purified from human plasma. Fibronectin increasesintegrin-mediated activation of latent TGFβ1 presented by LTBP1 and/orLTBP3.

FIG. 6 is a graph demonstrating that SR-AB1 is a context-independentinhibitor of TGFβ1 activation. SR-AB1 was shown to inhibitintegrin-dependent activation of TGFβ1 independent of the presentingmolecule.

FIGS. 7A, 7B, and 7C present data confirming LTBP-selective inhibitionof TGFβ1 large latent complex (LLC). FIG. 7A demonstrates that SR-AB2specifically binds LTBP-proTGFβ1 complex; it does not bind proTGFβ1 orLTBP1 alone. SR-AB2 also does not bind GARP-proTGFβ1. FIG. 7B depictsthat SR-AB2 inhibits integrin activation of LTBP1-proTGFβ1 (human andmouse complexes). FIG. 7C depicts that SR-AB2 inhibits integrinactivation of LTBP3-proTGFβ1.

FIG. 8 presents the heavy chain and light chain variable regionsequences of SR-AB2 (SEQ ID NOs: 7-8, respectively, in order ofappearance). Complementary determining regions (CDRs) are underlined.

FIG. 9 is a graph demonstrating the binding specificity of SR-AB2 toLTBP1-proTGFβ1 and LTBP3-proTGFβ1 complexes.

FIGS. 10A-10B provide data showing context-selective inhibition ofmatrix-associated TGFβ1 activation by SR-AB2. FIG. 10A demonstrates thatSR-AB2 inhibits LTBP-proTGFβ, wherein the transfected proTGFβ1 ispresented by endogenous LTBP1/3. FIG. 10B demonstrates that SR-AB2 doesnot inhibit GARP-presented TGFβ1 activation. These assays were performedin LN229 cells, which express high LTBP3 mRNA, low LTBP1 mRNA,undetectable GARP, and undetectable LRRC33. TGFβ activity, normalized tovehicle, is shown on the y-axis.

FIG. 11 presents binding profiles and affinity data for LTBPcomplex-specific antibodies SR-AB10, SR-AB2, and SR-13.

FIGS. 12A and 12B are graphs showing improved potency of optimized LTBPcomplex-specific antibodies. FIG. 12A provides a graph showing improvedinhibitory potency of SR-AB14 (an optimized SR-AB10) as measured bycell-based TGFβ reporter assays. FIG. 12B provides a graph showingimproved inhibitory potency of SR-AB15 (an optimized SR-AB13) asmeasured by cell-based TGFβ assays.

FIGS. 13A and 13B are graphs showing improved potency of optimized LTBPcomplex-specific antibodies after CDR-H3 mutagenesis (i.e., SR-AB20,SR-AB21, SR-AB22, and SR-AB23), as measured by cell-based TGFβ reporterassays.

FIGS. 14A and 14B are graphs showing improved potency of optimized LTBPcomplex-specific antibodies after CDR-H3 mutagenesis (i.e., SR-AB24,SR-AB25, SR-AB26, SR-AB27, SR-AB28, and SR-AB29), as measured bycell-based TGFβ reporter assays.

FIG. 15 is a graph that shows affinity matured antibodies show specificbinding to the LTBP-proTGFβ1 complex.

FIG. 16 is a graph showing improved potency of optimized LTBPcomplex-specific antibodies after cycles 1, 2 and 3 of antibodyoptimization as measured by cell-based TGFβ reporter assays.

FIG. 17 depicts the results of an enzyme-linked immunosorbent assay(ELISA) showing antibody binding to baculovirus (BV) particles, whichtests antibody polyspecificity.

FIG. 18 depicts the results of affinity-capture Self-interactionNanoparticle Spectroscopy (AC-SINS) assay, which tests antibodyself-interaction. Increased plasmon wavelength indicatesself-interaction.

FIG. 19 is a graph showing treatment with SR-AB42 and SR-AB31 inhibitedthe increase in hydroxyproline (HYP) (μg/mg tissue) in liver tissue inanimals on a choline-deficient high fat diet (CDHFD).

FIG. 20A is a graph showing relative ratios of phosphorylated versustotal (phosphorylated and unphospohrylated) Smad2/3 (pSMAD2/3:tSMAD2/3)in an Alport mouse model. A single dose of SR-AB42 or SR-AB63 wassufficient to significantly inhibit pSmad2/3 signaling in whole kidneylysates. FIG. 20B is a graph showing the amount of phosphorylatedSMAD2/3 (pSMAD2/3) as determined by ELISA, and FIG. 20C is a graphshowing the amount of total SMAD2/3 (tSMAD2/3) protein as determined byELISA. As shown by FIG. 20B and FIG. 20C, reduction of pSMAD iscontributing to the change in ratio shown in FIG. 20A.

FIG. 21 is a graph showing that the lead cycle 3 antibodies show noinhibition in the LTBP-TGFβ33 assay.

FIG. 22 provides 5 representative PSR-stained images from controls,CDHFD mice treated with Reference Ab, a TGFβ3 inhibitor, or both (left).A graph showing picosirius red area (%) in liver sections of CDHFD micetreated with Reference Ab, a TGFβ3 inhibitor, or both, as compared tocontrol, is also provided (right).

FIG. 23 shows LTBP-complex antibodies such as SR-AB63 are highlyspecific and have picomolar monovalent affinities.

FIG. 24 shows LTBP-complex antibodies such as SR-AB42 are highlyspecific and have picomolar monovalent affinities.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides compositions that are useful for reducingactivation of TGFβ. Inhibitors that target latent proTGFβ complexes,upstream of growth factor-receptor interaction, are generally referredto as activation inhibitors of TGFβ.

To date, four presenting molecules for TGFβ have been identified: latentTGF beta-binding protein 1 (“LTBP1”), latent TGF beta-binding protein 3(“LTBP3”), glycoprotein A repetitions predominant (“GARP”) andleucine-rich repeat-containing protein 33 (“LRRC33”). Each of thesepresenting molecules can form disulfide bonds with a homodimericpro-protein complex of the TGFβ precursor, i.e., proTGFβ. The proTGFβcomplex remains dormant (latent) in the respective extracellular niche(e.g., ECM and immune cell surface) until activation events trigger therelease of soluble growth factor from the complex.

As compared to the TGFβ growth factors and the receptors, which areexpressed broadly, the presenting molecules show more restricted orselective (e.g., tissue-specific) expression patterns, giving rise tofunctional compartmentalization of TGFβ activities by virtue ofassociation. The four presenting molecule-proTGFβ complexes, namely,LTBP1-proTGFβ, LTBP3-proTGFβ, GARP-proTGFβ and LRRC33-proTGFβ3,therefore, provide discrete “contexts” of TGFβ signaling within thetissue in which the presenting molecules are expressed. These contextsmay be divided into two broad categories: i) TGFβ signaling associatedwith the ECM (e.g., matrix-associated TGFβ function); and ii) TGFβsignaling associated with cells (particularly certain immune cellfunction). The LTBP1-proTGFβ3 and LTBP3-proTGFβ complexes fall under thefirst category, while GARP-proTGFβ and LRRC33-proTGFβ complexes fallunder the second category. Thus, disclosed herein are inhibitors of TGFβthat are capable of selectively inhibiting the activation of TGFβ thatis associated with the ECM. In some embodiments, the inhibitors are alsoselective for a particular TGFβ isoform (e.g., proTGFβ1, proTGFβ2,and/or proTGFβ33).

In exemplary embodiments, the compositions described herein are usefulfor selectively reducing activation of TGFβ1 in the context of an LTBPprotein, e.g., a LTBP1 and/or a LTBP3 protein. Such compositionsadvantageously inhibit activation of extracellular matrix-associatedTGFβ1, without inhibiting TGFβ1 in the context of the immune-associatedTGFβ1 presenting molecules GARP and LRRC33. The compositions describedherein are useful for treating disorders associated with TGFβ1activation, e.g., fibrotic disorders. Accordingly, in embodiments, theinvention provides compositions for reducing activation of TGFβ1,methods of use thereof, methods of manufacture, and treatment methods.Methods of selecting a TGFβ1 inhibitor for subjects exhibiting symptomsof a fibrotic disorder are also provided.

Definitions

In order that the disclosure may be more readily understood, certainterms are first defined. These definitions should be read in light ofthe remainder of the disclosure and as understood by a person ofordinary skill in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by a person of ordinary skill in the art. Additionaldefinitions are set forth throughout the detailed description.

Affinity: Affinity is the strength of binding of a molecule (such as anantibody) to its ligand (such as an antigen). It is typically measuredand reported by the equilibrium dissociation constant (K_(D)). K_(D) isthe ratio of the antibody dissociation rate (“off rate” or K_(off)), howquickly it dissociates from its antigen, to the antibody associationrate (“on rate” or K_(on)) of the antibody, how quickly it binds to itsantigen. For example, an antibody with an affinity of ≤1 μM has a K_(D)value that is 1 μM or lower (i.e., 1 μM or higher affinity) determinedby a suitable in vitro binding assay. Suitable in vitro assays, such asBiolayer Interferometry (e.g., Octet) or surface plasmon resonance(e.g., Biacore System) can be used to assess affinities, as measured byK_(D) values based on well-known methods.

Affinity maturation: Affinity maturation is a type of antibodyoptimization and is a process of improving the affinity of an antibodyor a fragment to its antigen and typically involves making one or morechanges to the amino acid sequence of the antibody or the fragment toachieve greater affinity. Typically, a parental antibody and anaffinity-matured counterpart retain the same epitope. Affinitymaturation may include diversification and/or mutagenesis of one or moreCDR sequences.

Antibody: The term “antibody” encompasses any naturally-occurring,recombinant, modified or engineered immunoglobulin orimmunoglobulin-like structure or antigen-binding fragment or portionthereof, or derivative thereof, as further described elsewhere herein.Thus, the term refers to an immunoglobulin molecule that specificallybinds to a target antigen, and includes, for instance, chimeric,humanized, fully human, and bispecific antibodies. Unless otherwisespecified to the contrary, the term “antibody” as used herein shallencompass antigen-binding fragments and varients thereof. An intactantibody will generally comprise at least two full-length heavy chainsand two full-length light chains, but in some instances can includefewer chains such as antibodies naturally occurring in camelids whichcan comprise only heavy chains. Antibodies can be derived solely from asingle source, or can be “chimeric,” that is, different portions of theantibody can be derived from two different antibodies. Antibodies, orantigen-binding portions thereof, can be produced in hybridomas, byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact antibodies. The term antibodies, as used herein, includesmonoclonal antibodies, bispecific antibodies, minibodies, domainantibodies, synthetic antibodies (sometimes referred to herein as“antibody mimetics”), chimeric antibodies, humanized antibodies, humanantibodies, antibody fusions (sometimes referred to herein as “antibodyconjugates”), respectively. In some embodiments, the term alsoencompasses peptibodies.

Antigen: The term “antigen” broadly includes any molecules comprising anantigenic determinant within a binding region(s) to which an antibody ora fragment specifically binds. An antigen can be a single-unit molecule(such as a protein monomer or a fragment) or a complex comprised ofmultiple components. An antigen provides an epitope, e.g., a molecule ora portion of a molecule, or a complex of molecules or portions ofmolecules, capable of being bound by a selective binding agent, such asan antigen-binding protein (including, e.g., an antibody). Thus, aselective binding agent may specifically bind to an antigen that isformed by two or more components in a complex. In some embodiments, theantigen is capable of being used in an animal to produce antibodiescapable of binding to that antigen. An antigen can possess one or moreepitopes that are capable of interacting with different antigen-bindingproteins, e.g., antibodies. In the context of the present disclosure, asuitable antigen is a complex (e.g., multimeric complex comprised ofmultiple components in association) containing a proTGF dimer (“smalllatent complex” or SLC) preferably in association with a presentingmolecule (together “large latent complex” or LLC). Each monomer of theproTGF dimer comprises a prodomain and a growth factor domain, separatedby a furin cleavage sequence. Two such monomers form the proTGF dimercomplex. This in turn is covalently associated with a presentingmolecule via disulfide bonds, which involve a cysteine residue presentnear the N-terminus of each of the proTGF monomer. This multi-complexformed by a proTGF dimer bound to a presenting molecule is generallyreferred to as a large latent complex. An antigen complex suitable forscreening antibodies or antigen-binding fragments, for example, includesa presenting molecule component of a large latent complex. Suchpresenting molecule component may be a full-length presenting moleculeor a fragment(s) thereof. Minimum required portions of the presentingmolecule typically contain at least 50 amino acids, but more preferablyat least 100 amino acids of the presenting molecule polypeptide, whichcomprises two cysteine residues capable of forming covalent bonds withthe proTGFβ1 dimer.

Antigen-binding portion/fragment: The terms “antigen-binding portion” or“antigen-binding fragment” of an antibody, as used herein, refers to oneor more fragments of an antibody that retain the ability to specificallybind to an antigen (e.g., LTBP1-proTGFβ1 and LTBP3-proTGFβ1).Antigen-binding portions include, but are not limited to, any naturallyoccurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. In some embodiments, an antigen-bindingportion of an antibody may be derived, e.g., from full antibodymolecules using any suitable standard techniques such as proteolyticdigestion or recombinant genetic engineering techniques involving themanipulation and expression of DNA encoding antibody variable andoptionally constant domains. Non-limiting examples of antigen-bindingportions include: (i) Fab fragments, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) F(ab′)2 fragments, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) Fd fragments consisting of the VH and CH1domains; (iv) Fv fragments consisting of the VL and VH domains of asingle arm of an antibody; (v) single-chain Fv (scFv) molecules (see,e.g., Bird et al. (1988) SCIENCE 242:423-426; and Huston et al. (1988)PROC. NAT′L. ACAD. SCI. USA 85:5879-5883); (vi) dAb fragments (see,e.g., Ward et al. (1989) NATURE 341: 544-546); and (vii) minimalrecognition units consisting of the amino acid residues that mimic thehypervariable region of an antibody (e.g., an isolated complementaritydetermining region (CDR)). Other forms of single chain antibodies, suchas diabodies are also encompassed. The term antigen-binding portion ofan antibody includes a “single chain Fab fragment” otherwise known as an“scFab,” comprising an antibody heavy chain variable domain (VH), anantibody constant domain 1 (CH1), an antibody light chain variabledomain (VL), an antibody light chain constant domain (CL) and a linker,wherein said antibody domains and said linker have one of the followingorders in N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b)VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL;and wherein said linker is a polypeptide of at least 30 amino acids,preferably between 32 and 50 amino acids.

Advanced fibrosis: As used herein, a subject suffers from advancedfibrosis if s/he has an advanced stage of a fibrotic disorder,particularly organ fibrosis, which renders the patient a candidate forreceiving, or in need of, an allograft transplant.

As needed: In the context of dosing regimens, the term “as needed”refers to a dosing regimen that is not based on a predetermined dosingschedule but instead based on one or more parameters or markers measuredor monitored periodically during treatment, which provides informationor guidance as to whether additional doses should be beneficial to thesubject/patient. For instance, a pharmaceutical composition comprising aTGFβ inhibitor such as TGFβ1/2/3 inhibitors (“pan” inhibitors), TGFβ1/2inhibitors and TGFβ1/3 inhibitors, may be administered, intermittently,on an “as needed” basis in a therapeutically effective amount sufficientto achieve and/or maintain clinical benefit (e.g., reduction of one ormore clinical markers of fibrosis). In some embodiments, administrationof a LTBP1/3-complex selective TGFβ inhibitor such as any one of theantibodies disclosed herein (e.g., Ab31, Ab34, Ab37, Ab38, Ab39, Ab40,Ab41, Ab42, Ab43, Ab44, Ab45, Ab62, Ab63, or Ab64 (optionally Ab42)) maybe used in combination with a method of determining or monitoringtherapeutic efficacy. In some embodiments, the LTBP1/3-complex selectiveTGFβ inhibitor is administered in patients only when clinical benefitfrom additional doses of the TGFβ inhibitor is expected. It iscontemplated that, in order to manage toxicities, intermittent or“as-needed” dosing regimen may be required more frequently withisoform-non-selective inhibitors of TGFβ, as compared to TGFβ1-selectiveinhibitors, such as those disclosed herein.

Bias: In the context of the present disclosure, the term “bias” refersto skewed or uneven affinity towards or against a subset of antigens towhich an antibody is capable of specifically binding. For example, anantibody is said to have bias when the affinity for one antigen complexand the affinity for another antigen complex are not equivalent (e.g.,more than five-fold difference in affinity). Antibodies characterized as“unbiased” have approximately equivalent affinities towards such antigencomplexes (e.g., less than five-fold difference in affinity). Antibodiesof the present disclosure “selectively” bind EMC-associated complexes(LTBP1-proTGFβ1 and LTBP3-proTGFβ). Such selective binding may in someembodiments comprise binding such that relative affinities between atleast one of the matrix-associated complexes and at least one(preferably both) of the cell-associated complexes (GARP-proTGFβ1 and/orLRRC33-proTGFβ1 complexes) is greater than fifty-fold.

Biolayer Interferometry (BLI): BLI is a label-free technology foroptically measuring biomolecular interactions, e.g., between a ligandimmobilized on the biosensor tip surface and an analyte in solution. BLIprovides the ability to monitor binding specificity, rates ofassociation and dissociation, or concentration, with precision andaccuracy. BLI platform instruments are commercially available, forexample, from ForteBio and are commonly referred to as the Octet®System. BLI can be employed in carrying out in vitro binding assays asdescribed herein.

Autoimmune disease: An autoimmune disease is a condition arising from anabnormal or overactive immune response to a normal body partImmunostimulating agents administered to such patients with autoimmuneconditions may exacerbate the condition.

Cell-associated proTGFβ1: The term refers to TGFβ1 or its signalingcomplex (e.g., pro/latent TGFβ1 ) that is membrane-bound (e.g., tetheredto cell surface). Typically, such cell is an immune cell. TGFβ1 that ispresented by GARP or LRRC33 is a cell-associated TGFβ1. GARP and LRRC33are transmembrane presenting molecules that are expressed on cellsurface of certain cells. GARP-proTGFβ1 and LRRC33-proTGFβ1 may becollectively referred to as “cell-associated” (or “cell-surface”)proTGFβ1 complexes, that mediate cell-associated (e g , immunecell-associated) TGFβ1 activation/signaling.

Chronic inflammation: In the context of the present disclosure, fibroticdisorders that involve chronic inflammation are characterized bycontinuous or persistent injury to a tissue such that it does notresolve in normal healing after an initial injury. Chronic inflammationrefers to a prolonged inflammatory response that involves a progressivechange in the type of cells present at the site of inflammation (e.g.,fibrotic tissues). It is characterized by the simultaneous destructionand repair of the tissue from the inflammatory process. It can follow anacute form of inflammation or be a prolonged low-grade form.

Clinical benefit: As used herein, the term “clinical benefits” isintended to include both efficacy and safety of a therapy. Thus,therapeutic treatment that achieves a desirable clinical benefit is bothefficacious and safe (e.g., with tolerable or acceptable toxicities oradverse events).

Combinatory or combinatorial epitope: A combinatorial epitope is anepitope that is recognized and bound by a combinatorial antibody at asite (i.e., antigenic determinant) formed by non-contiguous portions ofa component or components of an antigen, which, in a three-dimensionalstructure, come together in close proximity to form the epitope. Thus,antibodies of the invention may bind an epitope formed by two or morecomponents (e.g., portions or segments) of a pro/latent TGFβ1 complex. Acombinatory epitope may comprise amino acid residue(s) from a firstcomponent of the complex, and amino acid residue(s) from a secondcomponent of the complex, and so on. Each component may be of a singleprotein or of two or more proteins of an antigenic complex. Acombinatory epitope is formed with structural contributions from two ormore components (e.g., portions or segments, such as amino acidresidues) of an antigen or antigen complex.

Complementary determining region: As used herein, the term “CDR” refersto the complementarity determining region within antibody variablesequences. There are three CDRs in each of the variable regions of theheavy chain and the light chain, which are designated CDR1, CDR2 andCDR3, for each of the variable regions. The exact boundaries of theseCDRs have been defined differently according to different systems. Thesystem described by Kabat (Kabat et al. (1987; 1991) Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md.) not only provides an unambiguous residue numbering systemapplicable to any variable region of an antibody, but also providesprecise residue boundaries defining the three CDRs on each of the heavyand light chains. These CDRs may be referred to as Kabat CDRs.

Conformational epitope: A conformational epitope is an epitope that isrecognized and bound by a conformational antibody in a three-dimensionalconformation, but not in an unfolded peptide of the same amino acidsequence. A conformational epitope may be referred to as aconformation-specific epitope, conformation-dependent epitope, orconformation-sensitive epitope. A corresponding antibody or fragmentthereof that specifically binds such an epitope may be referred to asconformation-specific antibody, conformation-selective antibody, orconformation-dependent antibody. Binding of an antigen to aconformational epitope depends on the three-dimensional structure(conformation) of the antigen or antigen complex.

Context-specific: Context-specific (or context-selective) antibodies ofthe invention (as opposed to “context-independent” antibodies) arecapable of binding selectively to a subset, but not all, of proTGFβ1complexes associated with a particular biological context. For example,matrix-selective targeting enables specific inhibition of TGFβ1 functionassociated with the ECM. ECM-selective inhibition can be achieved by theuse of antibodies or fragments thereof that selectively target the ECMcomponents, LTBP1-proTGFβ1 and/or LTBP3-proTGFβ1. Antibodies andfragments disclosed herein therefore represent a class ofcontext-specific antibodies. LTBP1-specific and LTBP3-specificinhibitors of TGFβ1 activation are also context-specific antibodies.

Cross-block/cross-blocking: a first antibody or antigen-binding portionthereof and a second antibody or antigen-binding portion thereofcross-block with each other with respect to the same antigen, forexample, as assayed by as measured by Biolayer Interferometry (such asOctet) or surface plasmon resonance (such as Biacore System), usingstandard test conditions, e.g., according to the manufacturer'sinstructions (e.g., binding assayed at room temperature, —20-25° C.).The first antibody or fragment thereof and the second antibody orfragment thereof may have the same epitope; may have non-identical butoverlapping epitopes; or, may have separate (different) epitopes whichare in close proximity in a three-dimensional space, such that antibodybinding is cross-blocked via steric hindrance. “Cross-block” means thatbinding of the first antibody to an antigen prevents binding of thesecond antibody to the same antigen, and similarly, binding of thesecond antibody to an antigen prevents binding of the first antibody tothe same antigen.

Dissociation rate: The term dissociation rate as used herein has themeaning understood by the skilled artisan in the pertinent art (e.g.,antibody technology) as refers to a kinetics parameter measured by howfast/slow a ligand (e.g., antibody or fragment) dissociates from itsbinding target (e.g., antigen). Dissociation rate is also referred to asthe “off” rate (“k_(OFF)”). Relative on/off rates between an antibodyand its antigen (i.e., k_(ON) and k_(OFF)) determine the overallstrength of the interaction, or affinity, typically expressed as adissociation constant, or K_(D). Therefore, equivalent affinities (e.g.,K_(D) values) may be achieved by having fast association (high k_(ON)),slow dissociation (low k_(OFF)), or contribution from both factors.Monovalent interactions may be measured by the use of monovalentantigen-binding molecules/fragments, such as fAb (Fab), whilst divalentinteractions may be measured by the use of divalent antigen-bindingmolecules such as whole immunoglobulins (e.g., IgGs). Dissociaitonkinetics may be expressed in terms of dissociation half-time (sometimesreferred to as half binding time), or t½, defined as a duration of timeit takes for one half the number of antibody molecules (e.g., mAb, Fab,etc.) to dissociate from bound antigen. Thus, antibodies with slowdissociation rates have long dissociation half-time, and antibodies withfast dissociation rates have short dissociation half-time.

Dosage: As used herein, typical therapeutic dosage of an antibody of thepresent invention ranges between about 1-30 mg/kg per dose. A typicaldosing regimen may include once a week, every 2 weeks, every 3 weeks,every 4 weeks, once a month, every 6 weeks, etc.

ECM-associated (or “matrix-associated”) TGFβ1: The term refers to TGFβ1or its signaling complex (e.g., pro/latent TGFβ31) that is a componentof (e.g., deposited into) the extracellular matrix. TGFβ1 that ispresented by LTBP1 or LTBP3 is an ECM-associated TGFβ1.

Effective amount: An “effective amount” (or therapeutically effectiveamount) is a dosage or dosing regimen that achieves statisticallysignificant clinical benefits in a patient population.

Fibrotic disorder: The term “fibrosis” or “fibrotic condition/disorder”refers to the process or manifestation characterized by the pathologicalaccumulation of extracellular matrix (ECM) components, such ascollagens, within a tissue or organ. Fibrosis can include primaryfibrosis, as well as secondary fibrosis that are associated with adisease or disorder.

GARP-proTGFβ1: As used herein, the term “GARP-proTGFβ1” refers to aprotein complex comprising a pro-protein form or latent form of atransforming growth factor-β1 (TGFβ1 ) protein associated with aglycoprotein-A repetitions predominant protein (GARP) or fragment orvariant thereof. The proTGFβ1 homodimer is capable of forming covalentassociation with a single molecule of GARP via disulfide bonds. The term“GARP-TGFβ1” may be used interchangeably. GARP-proTGFβ1 expression islimited to certain cell types, such as regulatory T cells (Treg).

Human antibody: The term “human antibody,” as used herein, is intendedto include antibodies having variable and constant regions derived fromhuman germline immunoglobulin sequences. The human antibodies of thepresent disclosure may include amino acid residues not encoded by humangermline immunoglobulin sequences (e.g., mutations introduced by randomor site-specific mutagenesis in vitro or by somatic mutation in vivo),for example in the CDRs and in particular CDR3 (e.g., CDR-H3 or CDR-L3mutagenesis).

Humanized antibody: The term “humanized antibody” refers to antibodies,which comprise heavy and light chain variable region sequences from anon-human species (e.g., a mouse) but in which at least a portion of theVH and/or VL sequence has been altered to be more “human-like,” i.e.,more similar to human germline variable sequences. One type of humanizedantibody is a CDR-grafted antibody.

Immune suppression/immunosuppression: The term immunosuppression refersto suppression or reduction of the strength of the body's immune system.Patients who “benefit from immunosuppression” include those who haveadvanced stages of organ fibrosis and are candidates for, beingconsidered for, or have undergone transplantation.

Isoform-specific: The term “isoform specificity” refers to an agent'sability to discriminate one isoform over other structurally relatedisoforms (i.e., selectivity). An isoform-specific TGFβ inhibitor exertsits inhibitory activity towards one isoform of TGFβ but not the otherisoforms of TGFβ at a given concentration. For example, anisoform-specific TGFβ1 antibody selectively binds TGFβ1. ATGFβ1-specific inhibitor (antibody) preferentially targets (bindsthereby inhibits) the TGFβ1 isoform over TGFβ2 or TGFβ3 withsubstantially greater affinity. For example, the selectivity in thiscontext may refer to at least a 500-1000-fold difference in respectiveaffinities as measured by an in vitro binding assay such as Octet andBiacor. In some embodiments, the selectivity is such that the inhibitorwhen used at a dosage effective to inhibit TGFβ1 in vivo does notinhibit TGFβ2 and TGFβ3. Context-specific inhibitors of the presentdisclosure are also isoform-specific.

Isolated: An “isolated” antibody as used herein, refers to an antibodythat is substantially free of other antibodies having differentantigenic specificities. In some embodiments, an isolated antibody issubstantially free of other unintended cellular material and/orchemicals.

Long-term or chronic administration: As used herein, a therapeuticregimen that involves over six months of treatment is consideredlong-term. In some patient populations, long-term therapeutic regimensinvolve administration of a drug (such as context-selective TGFβ1inhibitors) for an indefinite duration of time.

LRRC33-proTGFfi1: As used herein, the term “LRRC33-TGFβ1 complex” refersto a complex between a pro-protein form or latent form of transforminggrowth factor-β1 (TGFβ1 ) protein and a Leucine-Rich Repeat-ContainingProtein 33 (LRRC33; also known as Negative Regulator Of Reactive OxygenSpecies or NRROS) or fragment or variant thereof. In some embodiments, aLRRC33-TGFβ1 complex comprises LRRC33 covalently linked with pro/latentTGFβ1 via one or more disulfide bonds. In other embodiments, aLRRC33-TGFβ1 complex comprises LRRC33 non-covalently linked withpro/latent TGFβ1. In some embodiments, a LRRC33-TGFβ1 complex is anaturally-occurring complex, for example a LRRC33-TGFβ1 complex in acell.

LTBP1-TGFβ1: As used herein, the term “LTBP1-TGFβ1 complex” (or“LTBP1-proTGFβ1 complex”) refers to a protein complex comprising apro-protein form or latent form of transforming growth factor-β1(TGFβ31) protein (may be referred to as “proTGFβ31” herein) and a latentTGF-beta binding protein 1 (LTBP1) or fragment or variant thereof. Insome embodiments, a LTBP1-TGFβ1 complex comprises LTBP1 covalentlylinked with pro/latent TGFβ1 via one or more disulfide bonds. In otherembodiments, a LTBP1-TGFβ1 complex comprises LTBP1 non-covalently linkedwith pro/latent TGFβ1. In some embodiments, a LTBP1-TGFβ1 complex is anaturally-occurring complex, for example a LTBP1-TGFβ1 complex in acell. An exemplary LTBP1-TGFβ1 complex is shown in FIG. 3.

LTBP3-TGFβ1: As used herein, the term “LTBP3-TGFβ1 complex” (or“LTBP3-proTGFβ1 complex”)refers to a protein complex comprising apro-protein form or latent form of transforming growth factor-β1(TGFβ31) protein (may be referred to as “proTGFβ31” herein) and a latentTGF-beta binding protein 3 (LTBP3) or fragment or variant thereof. Insome embodiments, a LTBP3-TGFβ1 complex comprises LTBP3 covalentlylinked with pro/latent TGFβ1 via one or more disulfide bonds. In otherembodiments, a LTBP3-TGFβ1 complex comprises LTBP1 non-covalently linkedwith pro/latent TGFβ1. In some embodiments, a LTBP3-TGFβ1 complex is anaturally-occurring complex, for example a LTBP3-TGFβ1 complex in acell. An exemplary LTBP3-TGFβ1 complex is shown in FIG. 3.

Macrophages: Macrophages are a type of white blood cells of the immunesystem and includes heterogeneous, phenotypically diverse subpopulationsof myeloid cells. Some macrophages differentiate from bonemarrow-derived, circulating monocytes, while others are tissue-specificmacrophages that reside within particular anatomical or tissue locations(“resident” macrophages). Tissue-specific macrophages include but arenot limited to: Adipose tissue macrophages; Kupffer cells (Liver); Sinushistiocytes (Lymph nodes); Alveolar macrophages (or dust cells,Pulmonary alveoli of lungs); Tissue macrophages (histiocytes) leading togiant cells (Connective tissue); Langerhans cells (Skin and mucosa);Microglia (Central nervous system); Hofbauer cells (Placenta);Intraglomerular mesangial cells (Kidney); Osteoclasts (Bone);Epithelioid cells (Granulomas); Red pulp macrophages (or Sinusoidallining cells, Red pulp of spleen); Peritoneal macrophages (Peritonealcavity); and, LysoMac (Peyer's patch). Macrophages, e.g., bone-marrowderived monocytes, can be activated by certain stimuli (such ascytokines) resulting in polarized phenotypes, e.g., M1 and M2. M2-biasedactivated macrophages are further classified into several phenotypicallydistinct subtypes, such as M2a, M2b, M2c (e.g., pro-fibrotic) and M2d(pro-tumor or TAM-like).

Matrix-associated proTGF LTBP1 and LTBP3 are presenting molecules thatare components of the extracellular matrix (ECM). LTBP1-proTGFβ1 andLTBP3-proTGFβ1 may be collectively referred to as “ECM-associated” (or“matrix-associated”) proTGFβ1 complexes, that mediate ECM-associatedTGFβ1 activation/signaling.

Maximally tolerated dose (MTD): The term MTD generally refers to, in thecontext of safety/toxicology considerations, the highest amount of atest article (such as a TGFβ1 inhibitor) evaluated with no observedadverse effect level (NOAEL). For example, the NOAEL for Ab2 in rats wasthe highest dose evaluated (100 mg/kg), suggesting that the MTD for Ab2is >100 mg/kg, based on a four-week toxicology study.

Myeloid-derived suppressor cell: Myeloid-derived suppressor cells(MDSCs) are a heterogeneous population of cells generated during variouspathologic conditions and thought to represent a pathologic state ofactivation of monocytes and relatively immature neutrophils. MDSCsinclude at least two categories of cells termed i) “granulocytic”(G-MDSC) or polymorphonuclear (PMN-MDSC), which are phenotypically andmorphologically similar to neutrophils; and ii) monocytic (M-MDSC) whichare phenotypically and morphologically similar to monocytes. MDSCs arecharacterized by a distinct set of genomic and biochemical features, andcan be distinguished by specific surface molecules. For example, humanG-MDSCs/PMN-MDSCs typically express the cell-surface markers CD11b,CD33, CD15 and CD66. In addition, human G-MDSCs/PMN-MDSCs may alsoexpress HLA-DR and/or Arginase. By comparison, human M-MDSCs typicallyexpress the cell surface markers CD11b, CD33 and CD14. The MDSCs mayalso express CD39 and CD73 to mediate adenosine signaling involved inorgan fibrosis (such as liver fibrosis, and lung fibrosis), cancer andmyelofibrosis). In addition, human M-MDSCs may also express HLA-DR. Inaddition to such cell-surface markers, MDSCs are characterized by theability to suppress immune cells, such as T cells, NK cells and B cellsImmune suppressive functions of MDSCs may include inhibition ofantigen-non-specific function and inhibition of antigen-specificfunction. MDSCs can express cell surface LRRC33 and/or LRRC33-proTGFβ1.

Myofibroblast: Myofibroblasts are cells with certain phenotypes offibroblasts and smooth muscle cells and generally express vimentin,alpha-smooth muscle actin (a-SMA; human gene ACTA2) and paladin. In manydisease conditions involving extracellular matrix dysregulations (suchas increased matrix stiffness), normal fibroblast cells becomede-differentiated into myofibroblasts in a TGFβ-dependent manner

Off rate (koFF): The off rate is a kinetic parameter of how fast or howslowly an antibody (such as mAb) or antigen-binding fragment (such asfAb) dissociates from its antigen and may be also referred to as thedissociation rate. Dissociation rates can be experimentally measured insuitable in vitro binding assays, such as BLI (Octet®)- and/or SPR(Biacore)-based systems. In the context of antibody-antigen bindingkinetics, the term “half-binding-time” (T½) or “dissociation half-time”refers to the duration of time required for half the number of antibodymolecules (e.g., mAb, Fab) to dissociate from the bound antigen (e.g.,LTBP1-proTGFβ1, LTBP3-proTGFβ31). Thus, an antibody that dissociatesslowly (i.e., low off rates) from its antigen has a long T½. Conversely,an antibody that dissociates rapidly (i.e., high off rates) from itsantigen has a short T½.

Pan-TGFβ inhibitor/pan-inhibition of TGFβ: The term “pan-TGFβ inhibitor”refers to any agent that is capable of inhibiting or antagonizing allthree isoforms of TGFβ. Such an inhibitor may be a small moleculeinhibitor of TGFβ isoforms. The term includes pan-TGFβ antibody whichrefers to any antibody capable of binding to each of TGFβ isoforms,i.e., TGFβ1, TGFβ2, and TGFβ3. In some embodiments, a pan-TGFβ antibodybinds and neutralizes activities of all three isoforms, i.e., TGFβ1,TGFβ2, and TGFβ3 activities.

Potency: The term “potency” as used herein refers to activity of a drug,such as a functional antibody (or fragment) having inhibitory activity,with respect to concentration or amount of the drug to produce a definedeffect. For example, an antibody capable of producing certain effects ata given dosage is more potent than another antibody that requires twicethe amount (dosage) to produce equivalent effects. Potency may bemeasured in cell-based assays, such as TGFβ activation/inhibitionassays. In some cases, the degree of TGFβ activation, such as activationtriggered by integrin binding, can be measured in the presence orabsence of test article (e.g., inhibitory antibodies) in a cell-basedsystem. Typically, antibodies with higher affinities tend to show higherpotency than antibodies with lower affinities.

Presenting molecule: Presenting molecules are proteins that formcovalent bonds with latent pro-proteins (e.g., proTGFβ31) and “present”the inactive complex in an extracellular niche (such as ECM or immunecell surface) thereby maintaining its latency until an activation eventoccurs. Known presenting molecules for proTGFβ1 include: LTBP1, LTBP3,GARP and LRRC33, which can form presenting molecule-proTGFβ1 complexes,namely, LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1 andLRRC33-proTGFβ1, respectively. LTBP1 and LTBP3 are components of theextracellular matrix (ECM); therefore, LTBP1-proTGFβ1 and LTBP3-proTGFβ1may be collectively referred to as “ECM-associated” (or“matrix-associated”) proTGFβ1 complexes, that mediate ECM-associatedTGFβ1 signaling/activities. GARP and LRRC33, on the other hand, aretransmembrane proteins expressed on cell surface of certain cells;therefore, GARP-proTGFβ1 and LRRC33-proTGFβ1 may be collectivelyreferred to as “cell-associated” (or “cell-surface”) proTGFβ1 complexes,that mediate cell-associated (e.g., immune cell-associated) TGFβ1signaling/activities.

ProTGFβ1: The term “proTGFβ1” as used herein is intended to encompassprecursor forms of inactive TGFβ1 complex that comprises a prodomainsequence of TGFβ1 within the complex. Thus, the term can include thepro-, as well as the latent-forms of TGFβ1. The expression “pro/latentTGFβ1 ” may be used interchangeably. The “pro” form of TGFβ1 existsprior to proteolytic cleavage at the furin site. Once cleaved, theresulting form is said to be the “latent” form of TGFβ1. The “latent”complex remains associated until further activation trigger, such asintegrin-driven activation event. The proTGFβ1 complex is comprised ofdimeric TGFβ1 pro-protein polypeptides, linked with disulfide bonds. Thelatent dimer complex is covalently linked to a single presentingmolecule via the cysteine residue at position 4 (Cys4) of each of theproTGFβ1 polypeptides. The adjective “latent” may be used generally todescribe the “inactive” state of TGFβ1, prior to integrin-mediated orother activation events. The proTGFβ1 polypeptide contains a prodomain(LAP) and a growth factor domain (SEQ ID NO: 12).

Regulatory T cell (Treg): “Regulatory T cells,” or Tregs, are a type ofimmune cells characterized by the expression of the biomarkers, CD4,forkhead box P3 (FOXP3), and CD25, as well as STAT5. Tregs are sometimesreferred to as suppressor T cells and represent a subpopulation of Tcells that modulate the immune system, maintain tolerance toself-antigens, and prevent autoimmune disease. Tregs areimmunosuppressive and generally suppress or downregulate induction andproliferation of effector T (Teff) cells. Tregs can develop in thethymus (so-called CD4+Foxp3+“natural” Tregs) or differentiate in theperiphery upon priming of naive CD4+T cells by antigen-presenting cells(APCs), for example, following exposure to TGFβ or retinoic acid. Tregcells produce and secrete cytokines including IL-10 and TGFβ1.Generally, differentiation of Treg and Th17 cells is negativelycorrelated.

Specific binding: As used herein, the term “specific binding” or“specifically binds” means that the interaction of the antibody, orantigen-binding portion thereof, with an antigen is dependent upon thepresence of a particular structure (e.g., an antigenic determinant orepitope). For example, the antibody, or antigen-binding portion thereof,binds to a specific protein rather than to proteins generally. In someembodiments, an antibody, or antigen-binding portion thereof,specifically binds to a target, e.g., TGFβ1, if the antibody has a K_(D)for the target of at least about 10⁻⁶ M. More preferably, the measuredK_(D) values of such antibody range between 10-100 nM. More preferably,the measured K_(D) values of such antibody range between 0.1-10 nM.

Subject: The term “subject” in the context of therapeutic applicationsrefers to an individual who receives clinical care or intervention, suchas treatment, diagnosis, etc. Suitable subjects include vertebrates,including but not limited to mammals (e.g., human and non-humanmammals). Where the subject is a human subject, the term “patient” maybe used interchangeably. In a clinical context, the term “a patientpopulation” or “patient subpopulation” is used to refer to a group ofindividuals that falls within a set of criteria, such as clinicalcriteria (e.g., disease presentations, disease stages, susceptibility tocertain conditions, responsiveness to therapy, etc.), medical history,health status, gender, age group, genetic criteria (e.g., carrier ofcertain mutation, polymorphism, gene duplications, DNA sequence repeats,etc.) and lifestyle factors (e.g., smoking, alcohol consumption,exercise, etc.).

TGF/1 inhibitor: The term “TGFβ inhibitor” refers to any agent capableof antagonizing biological activities or function of TGFβ growth factor(e.g., TGFβ31, TGFβ2 and/or TGFβ3 ). The term is not intended to limitits mechanism of action and includes, for example, neutralizinginhibitors, receptor antagonists, soluble ligand traps, and activationinhibitors of TGFβ.

T helper 17 cell: T helper 17 cells (Th17) are a subset ofpro-inflammatory T helper cells characterized by the markers STAT3 andRORyt and the production of cytokines including interleukin 17(IL-17A/F) and IL-22. Th17 cells are differentiated when naive T cellsare exposed to TGFβ and IL-6. Th17 cells are generally associated withtissue inflammation, autoimmunity and clearance of certain pathogens.The differentiation of Th17 cells and Treg cells is generally inverselyrelated. Imbalance in Th17-to-Treg ratios (e.g., “Th17/Treg”) has beenimplicated in a number of pathologies, such as fibrotic conditions andautoimmune conditions.

Th17/Treg ratio: Th17-to-Treg ratios refer to measured ratios (relativeproportions) of the number of Th17 cells versus the number of Treg cellsin a tissue or sample of interest. Typically, known cell markers areused to identify, sort or isolate the cell types. Such markers includecell-surface molecules expressed on the particular cell type; a cytokineor a panel of cytokines produced (e.g., secreted) by the particular celltype, and/or mRNA expression of certain gene markers that serve as asignature/profile of the particular cell type. For example, theTh17/Treg ratio of one (1) means that there is an equal or equivalentnumber of each of the cell types within the tissue or sample beingevaluated. The Th17/Treg ratio of two (2) means that there isapproximately twice the number of Th17 cells as compared to Treg cellsin the tissue or sample. An elevated Th17/Treg ratio may arise from anincreased number of Th17 cells, a decreased number of Treg cells, orcombination thereof.

Therapeutic window: The term “therapeutic window” refers to a range ofdoses/concentrations that produces therapeutic response without causingsignificant/observable/unacceptable adverse effect (e.g., within adverseeffects that are acceptable or tolerable) in subjects. Therapeuticwindow may be calculated as a ratio between minimum effectiveconcentrations (MEC) to the minimum toxic concentrations (MTC). Toillustrate, a TGFβ1 inhibitor that achieves in vivo efficacy at 10 mg/kgand shows tolerability or acceptable toxicities at 100 mg/kg provides atleast a 10-fold (e.g., 10x) therapeutic window. By contrast, apan-inhibitor of TGFβ that is efficacious at 10 mg/kg but causes adverseeffects at 5 mg/kg is said to have “dose-limiting toxicities.” Forexample, the applicants have found that a context-independent TGFβ1inhibitor antibody is efficacious at dosage ranging between about <3 and30 mg/kg/week and is free of observable toxicities associated withpan-inhibition of TGFβ at least 100 mg/kg/week for 4 weeks inpreclinical models such as rats. Based on this, the context-independentTGFβ1 inhibitor antibody shows at minimum a 3.3-fold and up to 33-foldtherapeutic window.

Toxicity: As used herein, the term “toxicity” or “toxicities” refers tounwanted in vivo effects in patients associated with a therapyadministered to the patients, such as undesirable side effects andadverse events. “Tolerability” refers to a level of toxicitiesassociated with a therapy or therapeutic regimen, which can bereasonably tolerated by patients, without discontinuing the therapy dueto the toxicities (i.e., acceptable level of toxicities). Typically,toxicity/toxicology studies are carried out in one or more preclinicalmodels prior to clinical development to assess safety profiles of a drugcandidate (e.g., monoclonal antibody therapy). Toxicity/toxicologystudies may help determine the “no observed adverse effect level(NOAEL)” and the “maximally tolerated dose (MTD)” of a test article,based on which a therapeutic window may be deduced. Preferably, aspecies that is shown to be sensitive to the particular interventionshould be chosen as a preclinical animal model in which safety/toxicitystudy is to be carried out. In case of TGFβ inhibition, suitable speciesinclude rats, dogs, and cynos. Mice are reported to be less sensitive topharmacological inhibition of TGFβ and may not reveal toxicities thatare potentially dangerous in other species, including human, althoughcertain studies report toxicities observed with pan-inhibition of TGFβin mice. To illustrate, the NOAEL for a context-independent TGFβ1inhibitor antibody in rats was the highest dose evaluated (100 mg/kg),suggesting that the MTD is >100 mg/kg per week, based on a four-weektoxicology study.

Treat/treatment: The term “treat” or “treatment” includes therapeutictreatments, prophylactic treatments, and applications in which onereduces the risk that a subject will develop a disorder or other riskfactor. Thus the term is intended to broadly mean: causing therapeuticbenefits in a patient by, for example, enhancing or boosting the body'simmunity; reducing or reversing immune suppression; reducing, removingor eradicating harmful cells or substances from the body; reducingdisease burden (e.g., tumor burden); preventing recurrence or relapse;prolonging a refractory period, and/or otherwise improving survival.Treatment does not require the complete curing of a disorder andencompasses embodiments in which one reduces symptoms or underlying riskfactors. In the context of combination therapy, the term may also referto: i) the ability of a second therapeutic to reduce the effectivedosage of a first therapeutic so as to reduce side effects and increasetolerability; ii) the ability of a second therapy to render the patientmore responsive to a first therapy; and/or iii) the ability toeffectuate additive or synergistic clinical benefits.

Variable region: The term “variable region” or “variable domain” refersto a portion of the light and/or heavy chains of an antibody, typicallyincluding approximately the amino-terminal 120 to 130 amino acids in theheavy chain and about 100 to 110 amino terminal amino acids in the lightchain. In certain embodiments, variable regions of different antibodiesdiffer extensively in amino acid sequence even among antibodies of thesame species. The variable region of an antibody typically determinesspecificity of a particular antibody for its target.

TGFβ1

In mammals, the transforming growth factor-beta (TGFβ3) superfamily iscomprised of at least 33 gene products. These include the bonemorphogenetic proteins (BMPs), activins, growth and differentiationfactors (GDFs), and the three isoforms of the TGFβ family: TGFβ1, TGFβ2,and TGFβ3. The TGFβ s are thought to play key roles in diverseprocesses, such as inhibition of cell proliferation, extracellularmatrix (ECM) remodeling, and immune homeostasis. The importance of TGFβ1for T cell homeostasis is demonstrated by the observation that TGFβ1−/−mice survive only 3-4 weeks, succumbing to multiorgan failure due tomassive immune activation (Kulkarni, A.B., et al., Proc Natl Acad SciUSA, 1993. 90(2): p. 770-4; Shull, M.M., et al., Nature, 1992.359(6397): p. 693-9). The roles of TGFβ2 and TGFβ3 are less clear.Whilst the three TGFβ isoforms have distinct temporal and spatialexpression patterns, they signal through the same receptors, TGFβRI andTGFβ3, although in some cases, for example for TGFβ2 signaling, type IIIreceptors such as betaglycan are also required (Feng, X.H. and R.Derynck, Annu Rev Cell Dev Biol, 2005. 21: p. 659-93; Massague, J., AnnuRev Biochem, 1998. 67: p. 753-91). Ligand-induced oligomerization ofTGFβRI/II triggers the phosphorylation of SMAD transcription factors,resulting in the transcription of target genes, such as Collal, Col3al,ACTA2, and SERPINE1 (Massague, J., J. Seoane, and D. Wotton, Genes Dev,2005. 19(23): p. 2783-810). SMAD-independent TGFβ signaling pathwayshave also been described, for example in cancer or in the aortic lesionsof Marfan mice (Derynck, R. and Y.E. Zhang, Nature, 2003. 425(6958): p.577-84; Holm, T.M., et al., Science, 2011. 332(6027): p. 358-61).

The biological importance of the TGFβ pathway in humans has beenvalidated by genetic diseases. Camurati-Engelman disease results in bonedysplasia due to an autosomal dominant mutation in the TGFB1 gene,leading to constitutive activation of TGFβ1 signaling (Janssens, K., etal., J Med Genet, 2006. 43(1): p. 1-11). Patients with Loeys/Dietzsyndrome carry autosomal dominant mutations in components of the TGFβsignaling pathway, which cause aortic aneurism, hypertelorism, and bifiduvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014.802: p. 95-105). As TGFβ pathway dysregulation has been implicated inmultiple diseases, several drugs that target the TGFβ pathway have beendeveloped and tested in patients, but with limited success. Most TGFβinhibitors described to date lack isoform specificity as brieflysummarized below.

Fresolimumab, a humanized monoclonal antibody that binds and inhibitsall three isoforms of TGFβ has been tested clinically in patients withfocal segmental glomerulosclerosis, malignant melanoma, renal cellcarcinoma, and systemic sclerosis (Rice, L. M., et al., J Clin Invest,2015. 125(7): p. 2795-807; Trachtman, H., et al., Kidney Int, 2011.79(11): p. 1236-43; Morris, J. C., et al., PLoS One, 2014. 9(3): p.e90353). Additional companies have developed monoclonal antibodiesagainst the TGFβ growth factors with varying degrees of selectivity forTGFβ isoforms. Such agents likely elicit toxicities in vivo throughresidual activity against other TGFβ family members besides TGFβ1. Thislack of isoform specificity may be due to the high degree of sequenceidentity between isoforms.

Other approaches to target the TGFβ pathway include ACE-1332, a solubleTGFβRII-Fc ligand trap from Acceleron (Yung, L. M., et al., A Am JRespir Crit Care Med, 2016. 194(9): p. 1140-1151), or small moleculeinhibitors of the ALKS kinase, such as Eli Lilly's galunisertib.ACE-1332 binds TGFβ1 and TGFβ3 with equally high affinity (Yung, L. M.,et al., Am J Respir Crit Care Med, 2016. 194(9): p. 1140-1151), and ALKSinhibitors block the activity of all growth factors that signal throughTGFR1. Substantial toxicities have been found in preclinical studiesusing ALKS inhibitors (Anderton, M. 6J., et al., Toxicol Pathol, 2011.39(6): p. 916-24; Stauber, A., et al., Clinical Toxicology, 2014. 4(3):p. 1-10), and sophisticated clinical dosing schemes are required tomaintain efficacy while reducing adverse events (Herbertz, S., et al.,Drug Des Devel Ther, 2015. 9: p. 4479-99). In fact, the question of TGFβsignaling specificity and its possible effect on toxicity observed withthe known TGFβ inhibitors has not been raised in most, if not all, ofthe candidate drugs that attempted to block TGFβ. For example, how muchof the toxicities are due to inhibition of TGFβ1 versus TGFβ2 and/orTGFβ3 has not been addressed. Similarly, modes of TGFβ activation havenot been taken into account in designing or developing ways toantagonize TGFβ signaling.

Recent structural insights into the activation mechanism of TGFβ1 (Shi,M., et al., Nature, 2011. 474(7351): p. 343-9) have enabled morespecific approaches to TGFβ inhibition (see, e.g., PCT/US2017/21972, theentire contents of which are incorporated herein by reference). Unlikeother cytokines, TGFβ superfamily members are not secreted as activegrowth factors, but as dimeric pro-proteins which consist of anN-terminal prodomain and a C-terminal growth factor domain. Cleavage ofproTGFβ1 by furin proteases separates the homodimeric growth factordomain from its prodomain, also referred to as latency associatedpeptide (LAP). However, the growth factor and LAP remain noncovalentlyassociated, forming a latent complex which is unable to bind itsreceptors and induce signaling. During translation, latent TGFβ1, alsocalled the small latent complex (SLC), becomes linked to “presentingmolecules” via disulfide bridges, forming the large latent complex(LLC). These molecules allow proTGFβ1 to be presented in specificcellular or tissue contexts. Two cysteines near the N-terminus of thelatent TGFβ1 link to appropriately positioned cysteines on thepresenting molecule. The identity of the presenting molecule depends onthe environment and cell type producing latent TGFβ1. For example,fibroblasts secrete latent TGFβ1 tethered to latent TGFβ-bindingproteins (LTBPs), which then associate with proteins in theextracellular matrix (ECM) (i.e., fibronectin, fibrillin-1) to linklatent TGFβ3 to the ECM (Robertson et al. Matrix Biol 47: 44-53 (2015)(FIG. 2A). On the surface of activated regulatory T cells latent TGFβ1is covalently linked to the transmembrane protein GARP (glycoprotein-Arepetitions predominant protein (GARP), and a protein closely related toGARP, LRRC33 (leucine-rich repeat-containing protein 33), serves as apresenting molecule for TGFβ1 on the surface of monocytes, macrophagesand microglia (Wang, R., et al., Mol Biol Cell, 2012. 23(6): p. 1129-39and T.A. Springer, Int. BMP Conference 2016).

A number of studies have shed light on the mechanisms of TGFβ1activation. Three integrins, αVβ6, αVβ8, and αVβ1 have been demonstratedto be key activators of latent TGFβ1 (Reed, N.I., et al., Sci TranslMed, 2015. 7(288): p. 288ra79; Travis, M.A. and D. Sheppard, Annu RevImmunol, 2014. 32: p. 51-82; Munger, J.S., et al., Cell, 1999. 96(3): p.319-28). aV integrins bind the RGD sequence present in TGFβ1 and TGFβ1LAPs with high affinity (Dong, X., et al., Nat Struct Mol Biol, 2014.21(12): p. 1091-6). Transgenic mice with a mutation in the TGFβ1 RGDsite that prevents integrin binding, but not secretion, phenocopy theTGFβ1−/− mouse (Yang, Z., et al., J Cell Biol, 2007. 176(6): p. 787-93).Mice that lack both 136 and 138 integrins recapitulate all essentialphenotypes of TGFβ1 and TGF133 knockout mice, including multiorganinflammation and cleft palate, confirming the essential role of thesetwo integrins for TGFβ1 activation in development and homeostasis(Aluwihare, P., et al., J Cell Sci, 2009. 122(Pt 2): p. 227-32). Key forintegrin-dependent activation of latent TGFβ1 is the covalent tether topresenting molecules; disruption of the disulfide bonds between GARP andTGFβ1 LAP by mutagenesis does not impair complex formation, butcompletely abolishes TGFβ1 activation by aV136 (Wang, R., et al., MolBiol Cell, 2012. 23(6): p. 1129-39). The recent structure of latentTGFβ1 illuminates how integrins enable release of active TGFβ1 from thelatent complex: the covalent link of latent TGFβ1 to its presentingmolecule anchors latent TGFβ1, either to the ECM through LTBPs, or tothe cytoskeleton through GARP or LRRC33. Integrin binding to the RGDsequence results in a force-dependent change in the structure of LAP,allowing active TGFβ1 to be released and bind nearby receptors (Shi, M.,et al., Nature, 2011. 474(7351): p. 343-9). The importance ofintegrin-dependent TGFβ1 activation in disease has also been wellvalidated. A small molecular inhibitor of aV131 protects againstbleomycin-induced lung fibrosis and carbon tetrachloride-induced liverfibrosis (Reed, N. I., et al., Sci Transl Med, 2015. 7(288): p.288ra79), and αVβ6 blockade with an antibody or loss of integrin 136expression suppresses bleomycin-induced lung fibrosis andradiation-induced fibrosis (Munger, J. S., et al., Cell, 1999. 96(3): p.319-28); Horan, G. S., et al., Am J Respir Crit Care Med, 2008. 177(1):p. 56-65). In addition to integrins, other mechanisms of TGFβ1activation have been implicated, including thrombospondin-1 andactivation by proteases such as matrix metalloproteinases (MMPs),cathepsin D or kallikrein. However, the majority of these studies wereperformed in vitro using purified proteins; there is less evidence forthe role of these molecules from in vivo studies. Knockout ofthrombospondin-1 recapitulates some aspects of the TGFβ1−/− phenotype insome tissues, but is not protective in bleomycin-induced lung fibrosis,known to be TGFβ3-dependent (Ezzie, M. E., et al., Am J Respir Cell MolBiol, 2011. 44(4): p. 556-61). Additionally, knockout of candidateproteases did not result in a TGFβ1 phenotype (Worthington, J. J., J. E.Klementowicz, and M.A. Travis, Trends Biochem Sci, 2011. 36(1): p.47-54). This could be explained by redundancies or by these mechanismsbeing critical in specific diseases rather than development andhomeostasis.

TGFβ3 has been implicated in a number of biological processes, includingfibrosis, immune-modulation and cancer progression. TGFβ1 was the firstidentified member of the TGFβ3 superfamily of proteins. Like othermembers of the TGFβ3 superfamily, TGFβ1 and the isoforms TGF132 andTGF133, are initially expressed as inactive precursor pro-protein forms(termed proTGF(3). TGFβ3 proteins (e.g., TGFβ1, TGF132 and TGF(33) areproteolytically cleaved by proprotein convertases (e.g., furin) to yieldthe latent form (termed latent TGF(3). In some embodiments, apro-protein form or latent form of a TGFβ3 protein (e.g., TGFβ1, TGF132and TGFβ3) may be referred to as “pro/latent TGFβ3 protein”. TGFβ1 maybe presented to other molecules in complex with multiple moleculesincluding, for example, GARP (to form a GARP-TGF(31 complex), LRRC33 (toform a LRRC33-TGF(31 complex), LTBP1 (to form a LTBP1-TGF(31 complex),and/or LTBP3 (to form a LTBP3-TGF(31 complex). The TGFβ1 present inthese complexes may be in either latent form (latent TGF(31) or inprecursor form (proTGF(31).

Isoform Selectivity and Mechanisms of Action of TGF inhibitors

From a safety standpoint, there has been an increasing recognition thatbroad inhibition of TGFβ across isoforms may be a cause of observedtoxicities, which underscores the fact that no TGFβ inhibitors have beensuccessfully developed to this day. To circumvent potentially dangerousadverse effects, a number of groups have recently turned to identifyinginhibitors that target a subset—but not all—of the isoforms and stillretain efficacy. From an efficacy standpoint, however, the prevailingview of the field remains to be that it is advantageous to inhibitmultiple isoforms of TGFβ3 to achieve therapeutic effects, and toaccommodate this, toxicity management by “careful dosing regimen” issuggested as a solution (Brennan et al. (2018) mAbs, 10:1, 1-17).Consistent with this premise, numerous groups are developing TGFβ3inhibitors that target more than one isoforms. These include lowmolecular weight antagonists of TGFβ3 receptors, e.g., ALKS antagonists,such as Galunisertib (LY2157299 monohydrate); monoclonal antibodies(such as neutralizing antibodies) that inhibit all three isoforms(“pan-inhibitor” antibodies) (see, for example, WO 2018/134681);monoclonal antibodies that preferentially inhibit two of the threeisoforms (e.g., antibodies against TGFβ1/2 (for example WO 2016/161410)and TGFβ1/3 (for example WO 2006/116002); and engineered molecules(e.g., fusion proteins) such as ligand traps (for example, WO2018/029367; WO 2018/129331 and WO 2018/158727). Similarly, inhibitorsof integrins such as αVβ6 also block integrin-dependent activation ofboth TGFβ1 and TGFβ3 and therefore may be considered asisoform-non-selective inhibitors of TGFβ signaling. In addition,examples of antibodies that selectively bind and neutralize both TGFβ1and TGFβ2 (i.e., TGFβ1/2 inhibitors) include XOMA 089 (or NIS793) andvariants (see, for example, WO 2016/161410).

Previously, Applicant demonstrated that inhibition of TGFβ1 alone wassufficient to sensitize immunosuppressive tumors to a checkpointinhibitor therapy even in tumors where both TGFβ1/3 are co-expressed(PCT/US2019/041373). Similarly, TGFβ1-selective inhibitors are shown tomitigate fibrosis in preclinical models, including mouse liver fibrosismodel where both the TGFβ1/3 isoforms are co-expressed in the fibrotictissue, albeit in discrete cell types, as observed byimmunohistochemistry (data now shown). Surprisingly, inhibition of TGFβ3promoted pro fibrotic phenotypes. The exacerbation of fibrosis isobserved when the TGFβ3 inhibitor is used alone. In addition, when usedin combination with a TGFβ1-selective inhibitor, the TGFβ3 inhibitorattenuated the anti-fibrotic effect of the TGFβ1-selective inhibitor, asevidenced by increased collagen accumulation in the fibrotic liver.These results raise the possibility that inhibitory potency againstTGFβ3 may be an undesirable feature of TGFβ inhibitors to be used astherapy in situations where fibrosis is a concern.

Beyond the fibrosis context, there is a broader implication to thisunexpected finding since the pro-fibrotic phenotype (e.g., increasedcollagen deposit into the ECM) is associated not only with fibrosis, butalso with aspects of cancer progression, such as tumor invasion andmetastasis. See, for example, Chakravarthy et al. (NatureCommunications, (2018) 9:4692. “TGF-β-associated extracellular matrixgenes link cancer-associated fibroblasts to immune evasion andimmunotherapy failure”). Diseased tissues with dysregulated ECM,including fibrotic tissues and stroma of various tumor types, canexpress both TGFβ1 and TGFβ3. As of today, multiple groups are makingeffort to develop TGFβ inhibitors that target both of these isoforms,such as ligand traps, neutralizing antibodies and integrin inhibitors.However, the finding presented herein cautions that such approach may infact exacerbate (e.g., worsen) the disease.

Accordingly, the present disclosure provides the teaching that for thetreatment of a disorder involving ECM dysregulation, such as fibrosisand cancer, a TGFβ inhibitor that does not specifically target TGFβ3should be selected. Preferably, such inhibitor is an isoform-selectiveinhibitor of TGFβ1, such as inhibitors that selectively targetLTBP1/3-associated TGFβ1 (e.g., as disclosed herein). Related methodsinclude a method for selecting a TGFβ inhibitor for use in the treatmentof a fibrotic disorder in a subject, wherein the method includes thesteps of: testing potency of one or more candidate inhibitors for theability to inhibit TGFβ1, TGFβ2 and TGFβ3, and selecting an inhibitorthat inhibits TGFβ1 but does not inhibit TGFβ3, for therapeutic use.Related treatment methods can further comprise a step of administeringto the subject the inhibitor that inhibits TGFβ1 but does not inhibitTGFβ3 in an amount sufficient to treat the fibrotic disorder or treat asubject having or at risk of developing a fibrotic disorder. Preferably,the selected inhibitor is an antibody or fragment thereof thatselectively inhibits LTBP1- and/or LTBP3-associated TGFβ1 signaling(e.g., as disclosed herein). In some embodiments, subjects at risk ofdeveloping a fibrotic disorder may suffer from a metabolic disorder,such as diabetes, obesity and NASH. The proposed exclusion of thesubpopulation of patients is aimed to reduce risk of triggering,facilitating or exacerbating a pro-fibrotic effect.

In addition to the possible concerns of inhibiting TGFβ3 addressedabove, Takahashi et al. (Nat Metab. 2019, 1(2): 291-303) recentlyreported a beneficial role of TGFβ2 in regulating metabolism. Theauthors identified TGFβ2 as an exercise-induced adipokine, whichstimulated glucose and fatty acid uptake in vitro, as well as tissueglucose uptake in vivo; which improved metabolism in obese mice; and,which reduced high fat diet-induced inflammation. Moreover, the authorsobesrved that lactate, a metabolite released from muscle duringexercise, stimulated TGFβ2 expression in human adipocytes and that alactate-lowering agent reduced circulating TGFβ2 levels and reducedexercise-stimulated improvements in glucose tolerance. Theseobservations suggest that therapeutic use of a TGFβ inhibitor withinhibitory activity towards the TGFβ2 isoform may be harmful at least inthe metabolic aspect.

Without being bound by particular theory, it is contemplated that it isadvantageous to select a TGFβ1-selective inhibitor as a TGFβ inhibitorfor use in the treatment of a metabolic disease, such as liver fibrosisassociated with NASH. In preferred embodiments, the TGFβ1-selectiveinhibitor selected for use in the treatment of the metabolic diseaseselectively inhibits LTBP1/3-associated TGFβ1, such as the antibodiesand fragments disclosed herein. Accordingly, the invention includes a aTGFβ inhibitor for use in the treatment of a metabolic disease in asubject, wherein the treatment comprises selection of a TGFβ inhibitorthat inhibits TGFβ1 but does not inhibit TGFβ2, optionally wherein theinhibitor is TGFβ1-selective, and administration of the inhibitor to asubject suffering from a metaboic disease. The metabolic disease may bea liver disease, such as liver fibrosis, NASH, NAFLD, optionallyaccompanied by obesity and/or type 2 diabetes. In preferred embodiments,the TGFβ1-selective inhibitor is an antibody or antigen-binding fragmentthereof that selectively targets matrix-associated TGFβ1 (e.g.,LTBP1-proTGFβ1 and LTBP3-proTGFβ31), such as those disclosed herein.

In preferred embodiments, a TGFβ inhibitor for use in the treatment of afibrotic disorder is an isoform-selective activation inhibitor of TGFβ1(such as the novel antibodies with low k_(OFF) or long t½ disclosedherein) capable of targeting matrix-associated TGFβ1-containing latentcomplexes in vivo.

The antibodies of the present disclosure work by preventing the step ofTGFβ1 activation. In some embodiments, such inhibitors can inhibitintegrin-dependent (e.g., mechanical or force-driven) activation ofTGFβ1. In some embodiments, such inhibitors can inhibitprotease-dependent or protease-induced activation of TGFβ1. The latterincludes inhibitors that inhibit the TGFβ1 activation step in anintegrin-independent manner In some embodiments, such inhibitors caninhibit TGFβ1 activation irrespective of the mode of activation, e.g.,inhibit both integrin-dependent activation and protease-dependentactivation of TGFβ1. Non-limiting examples of proteases which mayactivate TGFβ1 include serine proteases, such as Kallikreins,Chemotrypsin, Trypsin, Elastases, Plasmin, thrombin, as well as zincmetalloproteases (MMP family) such as MMP-2, MMP-9, MMP-12, MMP-13 andADAM proteases (e.g., ADAM10 and ADAM17) Kallikreins includeplasma-Kallikreins and tissue Kallikreins, such as KLK1, KLK2, KLK3,KLK4, KLKS, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14and KLK15.

Latent TGFβ-Binding Proteins (LTBPs)

In mammals there are four known LTBPs, LTBP1-4, each with multiplesplice variants (Robertson, I.B., et al., Matrix Biol, 2015. 47: p.44-53). LTBP2 is the only LTBP that does not associate with latent TGFβ(Saharinen, J. and J. Keski-Oja, Mol Biol Cell, 2000. 11(8): p.2691-704). While the association between LTBP1 or LTBP3 and latent TGFβ1has been well validated, the role of LTBP4 in TGFβ presentation is lessclear. The complex with LTBP4 and latent TGFβ1 appears to form much lessefficiently, potentially due to the absence of several negativelycharged residues in the TGFβ-binding domain of LTBP4 (Saharinen, J. andJ. Keski-Oja, Mol Biol Cell, 2000. 11(8): p. 2691-704; Chen, Y., et al.,J Mol Biol, 2005. 345(1): p. 175-86). Both LTBP4S−/− mice andUrban-Rifkin-Davis syndrome patients, who have null mutations in LTBP4,suffer from disrupted elastic fiber assembly (Urban, Z., et al., Am JHum Genet, 2009. 85(5): p. 593-605; Dabovic, B., et al., J Cell Physiol,2015. 230(1): p. 226-36). Additionally, while LTBP4S−/− mice have a lungseptation and an elastogenesis defect, transgenic mice with an LTBP4that cannot form a complex with latent TGFβ1 have no obvious phenotype(Dabovic, B., et al., J Cell Physiol, 2015. 230(1): p. 226-36). WhetherLTBP4 is directly involved in regulation of latent TGFβ1 by functioningas a presenting molecule is unclear; LTBP4 may instead be required forproper formation of elastic fibrils in the ECM and its loss indirectlyaffect latent TGFβ1 activation through defects in the ECM.

In one aspect, the present invention is directed to inhibitors, e.g.,immunoglobulins, e.g., antibodies, or antigen-binding portions thereof,that selectively bind to a complex containing a TGFβ pro-protein and aLTBP protein (e.g., LTBP1 or LTBP3). In a preferred embodiment, the TGFβprotein is TGFβ1. In some embodiments, the binding molecules disclosedherein bind selectively to a complex containing pro/latent TGFβ1 andLTBP1 or LTBP3. Such binding molecules can allow TGFβ1 activity to beselectively modulated in a context-dependent manner, i.e., by modulatingTGFβ1 in the context of a LTPB protein, without modulating the activityof TGFβ1 complexed with other presenting molecules (e.g., GARP and/orLRRC33).

Antibodies that Selectively Inhibit LTBP-Mediated TGFβ Activation

The present invention provides novel, TGFβ inhibitors that selectivelytarget matrix- or ECM-associated TGFβ activities. More specifically,such inhibitors include isoform-specific, context-selective inhibitorsof TGFβ1 activation that specifically bind latent forms of TGFβ1 (e.g.,proTGFβ1 complex) within the ECM environment and prevent release ofmature growth factor from the complex at the niche. Suchmatrix-targeting inhibitors are context-specific in that theyselectively bind proTGFβ1 associated with ECM presenting molecules,namely, LTBP1 and/or LTBP3. Thus, disclosed herein are monoclonalantibodies and fragments thereof capable of binding an epitope presentin an LTBP1-proTGFβ1 complex and/or LTBP3-proTGFβ1 complex, whereas theepitope is not present in a GARP-proTGFβ1 complex and/or LRRC33-proTGFβ1complex.

In some embodiments, the context-selective inhibitors of the presentdisclosure are capable of specifically binding both a humanLTBP1-proTGFβ1 complex and a human LTBP3-proTGFβ1 complex, withaffinities of <5 nM each (measured K_(D) values) in a suitable in vitrobinding assay, such as Octet. In preferred embodiments, such antibodiesbind both a human LTBP1-proTGFβ1 complex and a human LTBP3-proTGFβ1 withaffinities of <5 nM each (measured K_(D) values) in a suitable in vitrobinding assay, such as Octet. On the other hand, these context-specificantibodies do not show any detectable binding to a human GARP-proTGFβ1complex or a human LRRC33-proTGFβ1 complex under the same assayconditions. Preferably, such antibody or the fragment binds each of thehuman LTBP1-proTGFβ1 complex and the human LTBP3-proTGFβ1 complex withKD of less than 1 nM.

In some embodiments, the context-selective inhibitors of the presentdisclosure are capable of specifically binding either a humanLTBP1-proTGFβ1 complex or a human LTBP3-proTGFβ1 complex. Neither showsany detectable binding to a human GARP-proTGFβ1 complex or a humanLRRC33-proTGFβ1 complex under the same assay conditions.

The art is familiar with suitable in vitro binding assays, including,for example, BLI-based assays such as Octet and SPR-based assays such asBiacore, which can be used to measure antibody-antigen interactions(e.g., binding kinetics). As used herein, “no binding” in these contextsmay refer to no detectable binding by a particular assay, e.g., thebinding, if any, is below the sensitivity of the assay. In someembodiments, “no binding” may refer to “no meaningful binding”, set by acutoff, which defines the minimum level required to be consideredmeaningful as measured by a particular assay system. For example, in aBLI (Octet) assay, 0.1 nm of optical shift measured at predeterminedanalyte (e.g., target antigen) concentrations (e.g., 100 nM, 200 nM,etc.) may indicate meaningful binding, below which may be considered asno binding (see for example, Example 9). Similarly, in a typical SPR(Biacore) assay, the cutoff level may be 0.2 RU (resonance unit). Insome embodiments, the signal at 0 nM antibody (e.g., background noise)is subtracted from all the sensorgrams obtained at higherconcentrations. Under these conditions, using SPR (Biacore), 0.2 RUs maybe a suitable cutoff.

In some embodiments, the context-selective inhibitors of the presentdisclosure are capable of specifically binding a human LTBP1-proTGFβ1complex or a human LTBP3-proTGFβ1 complex without showing detectablebinding to a human GARP-proTGFβ1 complex, as measured by BLI, under thesame assay conditions as used to measure binding to human LTBP1-proTGFβ1complex and/or human LTBP3-TGFβ1.

In some embodiments, the context-selective inhibitors of the presentdisclosure bind a human LTBP1-proTGFβ1 complex or a human LTBP3-proTGFβ1complex with a K_(D) that is at least 50 times lower (e.g., at least 75times lower, at least 100 times lower) than the K_(D) when binding to ahuman GARP-proTGFβ1 complex under the same assay conditions. In someembodiments, the K_(D) is as determined by BLI or SPR. In someembodiments, the K_(D) is as determined by SPR.

In some embodiments, the context-selective inhibitors of the presentdisclosure are capable of specifically binding a human LTBP1-proTGFβ1complex or a human LTBP3-proTGFβ1 complex without showing detectablebinding to an LRRC33-proTGFβ1 latent complex, as measured by BLI, underthe same assay conditions as used to measure binding to humanLTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1.

In some embodiments, the context-selective inhibitors of the presentdisclosure bind a human LTBP1-proTGFβ1 complex or a human LTBP3-TGFβ1complex with a K_(D) that is at least 50 times lower (e.g., at least 75times lower, at least 100 times lower) than the K_(D) when binding to ahuman LRRC33-proTGFβ1 complex under the same assay conditions. In someembodiments, the K_(D) is as determined by BLI or SPR. In someembodiments, the K_(D) is as determined by SPR.

The invention includes the recognition that preferred antibodies (e g,immunoglobulins and antigen-binding fragments such as Fabs, as well asengineered constructs incorporating such fragments), once bound to itstarget/antigen (e.g., human LTBP1-proTGFβ1, human LTBP3-TGFβ31),dissociates slowly from the antigen. Thus, the novel antibodies of theinstant invention are selected not only for their high overallaffinities (such as KD of no more than 5 nM) but especially for theirlow dissociation rates. According to the present disclosure, suchantibodies have dissociation rates of <5×10 (1/s) , as measured by BLI(e.g., when binding to human LTBP1-proTGFβ1 and/or human LTBP3-TGFβ31).Such dissociation rates of of <5×10⁻⁴ (1/s) of the antibodies orantigen-binding fragments may be monovalent dissociation rates ordivalent dissociation rates. In some embodiments, the antibody or thefragment dissociates slowly from human LTBP1-proTGFβ1 and/or humanLTBP3-TGFβ1, preferably for both human LTBP1-proTGFβ1 and humanLTBP3-TGFβ1, with a monovalent dissociation half-time (t½) of at least45 minutes (e.g., >45, 60, 75, 90 minutes) as measured by SPR. On theother hand, should binding to a human GARP-proTGFβ1 and/or LRRC33-TGFβ1complex be detectable, such antibody dissociates rapidly from a humanGARP-proTGFβ1 and/or human LRRC33-TGFβ1 complex(es), particularly thehuman GARP-proTGFβ1. In some embodiments, the antibody dissociates fromhuman GARP-proTGFβ1 with t′ of less than 5 minutes as measured by SPR.In particularly preferred embodiments, the antibody or the fragmentsshow species cross-reactivity such that they bind murine counterpartswith equivalent affinities.

The TGFβ1 present in these complexes may be in either latent form(latent TGFβ31) or in precursor form (proTGFβ31). In one embodiment, theinhibitors do not significantly bind to LTBP1 alone (e.g., when notcomplexed with TGFβ31). In another embodiment, the inhibitors do notsignificantly bind to LTBP3 alone (e.g., when not complexed withTGFβ31). In another embodiment, the inhibitors do not significantly bindto TGFβ1 alone (e.g., pro or latent TGFβ1 not complexed with LTBP1 orLTBP3, or mature TGFβ1 ). In another embodiment, the inhibitors thatselectively bind a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex donot significantly bind to a complex containing TGFβ1 and anotherpresenting molecule, e.g., a GARP-TGFβ1 complex (e.g., GARP complexed topro- or latent TGFβ1 ) and/or a LRRC33-TGFβ1 complex (e.g., LRRC33complexed to pro- or latent TGFβ31). In one embodiment, the inhibitorsthat selectively bind LTBP1/3-TGFβ1 do not significantly bind one ormore (e.g., two or more, three or more, or all four) of the following:LTBP1 alone, TGFβ1 alone, a GARP-TGFβ1 complex, and a LRRC33-TGFβ1complex. In addition, in some embodiments, the inhibitors do notsignificantly bind LTBP3 alone.

As used herein, the term “inhibitor” refers to any agent capable ofblocking or antagonizing TGFβ1 signaling. Such agents may include smallmolecule antagonists of TGFβ1 and biologic antagonists of TGFβ1 (e.g.,protein fragments and antibodies). In some embodiments, the inhibitormay be an antibody (including fragments thereof, such as DomainAntibodies (dAbs) as described in, for example, U.S. Pat. Nos.6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245), a smallmolecule inhibitor, an Adnectin, an Affibody, a DARPin, an Anticalin, anAvimer, a Versabody or a gene therapy. Use of inhibitors encompassed bythe present invention also includes antibody mimetics, such asmonobodies and single-domain antibodies. Monobodies are syntheticbinding proteins that typically employ a fibronectin type III domain(FN3) as a molecular scaffold. Monobodies include AdnectinsTM which arebased on the 10th fibronectin type III domain.

In some aspects, the inhibitors, e.g., antibodies, or antigen-bindingportions thereof, selectively bind to an epitope present on aLTBP1/3-TGFβ1 complex, that is not present on a GARP-TGFβ1 complexand/or a LRRC33-TGFβ1 complex. In some embodiments, the epitope isavailable due to a conformational change in LTBP1/3 and/or TGFβ1 thatoccurs when LTBP1/3 and TGFβ1 form a complex. In this embodiment, theepitope is not present in LTBP1/3 or TGFβ1 when the proteins are notassociated in a complex. In one embodiment, the epitope is present onTGFβ1, when TGFβ1 is in a complex with LTBP1 or LTBP3. In anotherembodiment, the epitope is present on LTBP1, when LTBP1 is in a complexwith TGFβ1. In another embodiment, the epitope is present on LTBP3, whenLTBP3 is in a complex with TGFβ1. In another embodiment, the epitopecomprises residues from both LTBP1 and TGFβ1. In another embodiment, theepitope comprises residues from both LTBP3 and TGFβ1.

Surprisingly, some of the LTBP1/3 complex-selective antibodies dislosedherein (e.g., Ab14, Ab20, Ab21-23, Ab17, and Ab24-29) are capable ofbinding to the small latent complex (proTGFβ1 C4S) in the absence of apresenting molecule (e.g., LTBP1/3) and yet exert context-selectivity.Without wishing to be bound by theory, this finding suggests that theantibodies (and variants thereof, or cross-competing antibodies) maybind an epitope that is available in the LTBP1/3-proTGFβ1 complex and inproTGFβ1 alone, but which is not available when an LRRC-type ofpresenting molecule (GARP or LRRC33) is present. The epitope might beentirely on latent TGFβ1, but gets occluded (directly or indirectly)when GARP or LRRC33 is complexed.

Alternatively, LTBP-selective inhibitors according to the presentdisclosure may bind a combinatorial epitope that comprises one or moreamino acid residues of LTBP1 or LTBP3 and one or more amino acidresidues of proTGFβ1, which confer the context-selectivity towards anLTBP-bound complex over GARP/LRRC33-bound complex. In these embodiments,selectivity towards the isoform (TGFβ31) as well as the context (ECM) isattributable to the combined contributions from both elements of theantigen complex.

In some embodiments, the inhibitors, e.g., antibodies, orantigen-binding portions thereof, are selective for the TGFβ1 isoform.In such embodiments, the inhibitors, e.g., antibodies, orantigen-binding portions thereof, do not bind to TGFβ2 and/or TGFβ3. Forexample, in one embodiment, the inhibitors, e.g., antibodies, orantigen-binding portions thereof, selectively bind a LTBP1/3-TGFβ1complex, but do not bind TGFβ2, or a complex containing TGFβ2. Inanother embodiment, the inhibitors, e.g., antibodies, or antigen-bindingportions thereof, selectively bind a LTBP1/3-TGFβ1 complex, but do notbind TGFβ3, or a complex containing TGFβ3.

In some embodiments, the inhibitors, e.g., antibodies, orantigen-binding portions thereof, do not prevent TGFβ1 from binding tointegrin. For example, in some embodiments, the inhibitors, e.g.,antibodies, or antigen-binding portions thereof, do not mask theintegrin-binding site of TGFβ1.

In one aspect, the invention provides functional inhibitors, e.g.,antibodies, that modulate TGFβ1 activity. In exemplary embodiments, theantibodies described herein are inhibitory antibodies, which inhibit thefunction or activity of TGFβ1. In some embodiments, the antibodies, orantigen-binding portions thereof, inhibit the activation (release) ofTGFβ1 from a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex. Thepresent disclosure provides, in exemplary embodiments,“context-specific” or “context-selective” inhibitors of TGFβ1activation. Such inhibitors can bind a LTBP1/3-TGFβ1 complex and inhibitactivation of TGFβ1 that is presented by LTBP1 or LTBP3, withoutinhibiting the activation of TGFβ1 presented by GARP and/or LRRC33.Accordingly, in some embodiments, the antibodies, or antigen-bindingportions thereof, described herein inhibit the release of mature TGFβ1from a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, but do notinhibit the release of mature TGFβ1 from a GARP-TGFβ1 complex and/or aLRRC33-TGFβ1 complex. Due to the differential localization of LTBP,GARP, and LRRC33, the context-specific inhibitors of TGFβ1 provided bythe present invention can block a particular subset of TGFβ1 activity invivo. In one embodiment, the context-specific antibodies provided hereinthat inhibit LTBP1/3-TGFβ1 but do not inhibit GARP-TGFβ1 or LRRC33-TGFβ1can be used to inhibit TGFβ1 localized to the extracellular matrix. Inanother embodiment, the context-specific antibodies can inhibit TGFβ1without modulating TGFβ1-associated immune activity or immune response.In another embodiment, the context-specific antibodies can be used toinhibit TGFβ1 activity associated with the extracellular matrix withoutmodulating TGFβ1 activity associated with hematopoietic cells.Accordingly, the context-specific antibodies can be used to inhibitLTBP1/3-associated TGFβ1 activity in applications in which TGFβ1activation in the context of GARP and/or LRRC33 is undesirable, asdescribed herein.

In some embodiments, the TGFβ1 comprises a naturally occurring mammalianamino acid sequence. In some embodiment, the TGFβ1 comprises a naturallyoccurring human amino acid sequence. In some embodiments, the TGFβ1comprises a human, a monkey, a rat or a mouse amino acid sequence.

In some embodiments, an antibody, or antigen-binding portion thereof,described herein selectively binds to a complex comprising a TGFβ1protein comprising the amino acid sequence set forth in SEQ ID NO: 9,and LTBP1 or LTBP3. In some embodiments, an antibody, or antigen-bindingportion thereof, described herein selectively binds to a LTBP1/3-TGFβ1complex which comprises a non-naturally-occurring TGFβ1 amino acidsequence (otherwise referred to herein as a non-naturally-occurringTGFβ1 ). For example, a non-naturally-occurring TGFβ1 may comprise oneor more recombinantly generated mutations relative to anaturally-occurring TGFβ1 amino acid sequence.

In some embodiments, an antibody, or antigen-binding portion thereof,described herein does not bind TGFβ2 and/or TGFβ3, or to proteincomplexes containing TGFβ2 and/or TGFβ3. Exemplary TGFβ2 and TGFβ3 aminoacid sequences are set forth in SEQ ID NOs: 10 and 11, respectively. Insome embodiments, a TGFβ1, TGFβ2, or TGFβ3 amino acid sequence comprisesan amino acid sequence as set forth in SEQ ID NOs: 12-23, as shown inTable 1. In some embodiments, a TGFβ1 amino acid sequence comprises anamino acid sequence as set forth in SEQ ID NOs: 24-31, as shown in Table2.

TGFβ1 (SEQ ID NO: 9) LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGR KPKVEQLSNMIVRSCKCSTGFβ2 (SEQ ID NO: 10) SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSC KCS TGFβ3(SEQ ID NO: 11) SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS

TABLE 1 Exemplary TGFβ1, TGFβ2, and TGFβ3 amino acid sequences ProteinSequence SEQ ID NO proTGFβ1LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDR 12VAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVE QLSNMIVRSCKCSproTGFβ1 C4S LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDR13 VAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVE QLSNMIVRSCKCSproTGFβ 1D2G LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDR14 VAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHGALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCSproTGFβ1 C4S D2GLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDR 15VAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHGALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCSproTGFβ2 SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDL 16LQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS proTGFβ2 C5SSLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDL 17LQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS proTGFβ 2C5S D2GSLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDL 18LQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKGALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS proTGFβ2 D2GSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDL 19LQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKGALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS proTGFβ3SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRE 20LLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS proTGFβ3 C7SSLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRE 21LLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS proTGFβ3 C7S D2GSLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRE 22LLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKGALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS proTGFβ3 D2GSLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRE 23LLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKGALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS

TABLE 2 Exemplary non-human TGFβ1 amino acid sequences Protein SpeciesSequence SEQ ID NO proTGFβ1 MouseLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYN 24STRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGFβ1 CynoLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYN 25STRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS TGFβ1 LAP MouseLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYN 26 C4SSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRR TGFβ1 LAP CynoLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYN 27 C4SSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRR proTGFβ1 MouseLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYN 28 C4S D2GSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHGALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGFβ1 MouseLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYN 29 C4SSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGFβ1 CynoLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYN 30 C4SSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGFβ1 CynoLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYN 31 C4S D2GSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHGALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

In some embodiments, an antibody, or antigen-binding portion thereof, asdescribed herein, is capable of selectively binding to an LTBP-TGFβ1complex. In some embodiments, antigenic protein complexes (e.g., aLTBP-TGFβ1 complex) may comprise an LTBP protein selected from thefollowing: LTBP1, LTBP2, LTBP3, and LTBP4.

In some embodiments, the antibody, or antigen-binding portion thereof,selectively binds an LTBP1-TGFβ1 complex. In some embodiments, the LTBP1protein is a naturally-occurring protein. In some embodiments, the LTBP1protein is a non-naturally occurring protein. In some embodiments, theLTBP1 protein is a recombinant protein. Such recombinant LTBP1 proteinmay comprise LTBP1, alternatively spliced variants thereof, and/orfragments thereof. Recombinant LTBP1 proteins may also be modified tocomprise one or more detectable labels. In some embodiments, the LTBP1protein comprises a leader sequence (e.g., a native or non-native leadersequence). In some embodiments, the LTBP1 protein does not comprise aleader sequence (i.e., the leader sequence has been processed orcleaved). Such detectable labels may include, but are not limited tobiotin labels, polyhistidine tags, myc tags, HA tags and/or fluorescenttags. In some embodiments, the LTBP1 protein is a mammalian LTBP1protein. In some embodiments, the LTBP1 protein is a human, a monkey, amouse, or a rat LTBP1 protein. In some embodiments, the LTBP1 proteincomprises an amino acid sequence as set forth in SEQ ID NO: 32 in Table3. In some embodiments, the LTBP1 protein comprises an amino acidsequence as set forth in SEQ ID NOs: 33 or SEQ ID NO: 34 in Table 3.

In some embodiments, an antibody, or antigen-binding portion thereof, asdescribed herein, is capable of binding to a LTBP3-TGFβ1 complex. Insome embodiments, the LTBP3 protein is a naturally-occurring protein. Insome embodiments, the LTBP3 protein is a non-naturally occurringprotein. In some embodiments, the LTBP3 protein is a recombinantprotein. Such recombinant LTBP3 protein may comprise LTBP3,alternatively spliced variants thereof and/or fragments thereof. In someembodiments, the LTBP3 protein comprises a leader sequence (e.g., anative or non-native leader sequence). In some embodiments, the LTBP3protein does not comprise a leader sequence (i.e., the leader sequencehas been processed or cleaved). Recombinant LTBP3 proteins may also bemodified to comprise one or more detectable labels. Such detectablelabels may include, but are not limited to biotin labels, polyhistidinetags, myc tags, HA tags and/or fluorescent tags. In some embodiments,the LTBP3 protein is a mammalian LTBP3 protein. In some embodiments, theLTBP3 protein is a human, a monkey, a mouse, or a rat LTBP3 protein. Insome embodiments, the LTBP3 protein comprises an amino acid sequence asset forth in SEQ ID NO: 35. In some embodiments, the LTBP3 proteincomprises an amino acid sequence as set forth in SEQ ID NOs: 36 or 37.

TABLE 3 [xemplary LTBP amino acid sequences. Protein Species SequenceSEQ ID NO LTBP1S HumanNHTGRIKVVFTPSICKVTCTKGSCQNSCEKGNTTTLISENGHAADTLTATNFR  32VVICHLPCMNGGQCSSRDKCQCPPNFTGKLCQIPVHGASVPKLYQHSQQPGKALGTHVIHSTHTLPLTVTSQQGVKVKFPPNIVNIHVKHPPEASVQIHQVSRIDGPTGQKTKEAQPGQSQVSYQGLPVQKTQTIHSTYSHQQVIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSKQEDCCGTVGTSWGFNKCQKCPKKPSYHGYNQMMECLPGYKRVNNTFCQDINECQLQGVCPNGECLNTMGSYRCTCKIGFGPDPTFSSCVPDPPVISEEKGPCYRLVSSGRQCMHPLSVHLTKQLCCCSVGKAWGPHCEKCPLPGTAAFKEICPGGMGYTVSGVHRRRPIHHHVGKGPVFVKPKNTQPVAKSTHPPPLPAKEEPVEALTFSREHGPGVAEPEVATAPPEKEIPSLDQEKTKLEPGQPQLSPGISTIHLHPQFPVVIEKTSPPVPVEVAPEASTSSASQVIAPTQVTEINECTVNPDICGAGHCINLPVRYTCICYEGYRFSEQQRKCVDIDECTQVQHLCSQGRCENTEGSFLCICPAGFMASEEGTNCIDVDECLRPDVCGEGHCVNTVGAFRCEYCDSGYRMTQRGRCEDIDECLNPSTCPDEQCVNSPGSYQCVPCTEGFRGWNGQCLDVDECLEPNVCANGDCSNLEGSYMCSCHKGYTRTPDHKHCRDIDECQQGNLCVNGQCKNTEGSFRCTCGQGYQLSAAKDQCEDIDECQHRHLCAHGQCRNTEGSFQCVCDQGYRASGLGDHCEDINECLEDKSVCQRGDCINTAGSYDCTCPDGFQLDDNKTCQDINECEHPGLCGPQGECLNTEGSFHCVCQQGFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYQGFQAPQDGQGCVDVNECELLSGVCGEAFCENVEGSFLCVCADENQEYSPMTGQCRSRTSTDLDVDVDQPKEEKKECYYNLNDASLCDNVLAPNVTKQECCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGFVPAGESSSEAGGENYKDADECLLFGQEICKNGFCLNTRPGYECYCKQGTYYDPVKLQCFDMDECQDPSSCIDGQCVNTEGSYNCFCTHPMVLDASEKRCIRPAESNEQIEETDVYQDLCWEHLSDEYVCSRPLVGKQTTYTECCCLYGEAWGMQCALCPLKDSDDYAQLCNIPVTGRRQPYGRDALVDFSEQYTPEADPYFIQDRFLNSFEELQAEECGILNGCENGRCVRVQEGYTCDCFDGYHLDTAKMTCVDVNECDELNNRMSLCKNAKCINTDGSYKCLCLPGYVPSDKPNYCTPLNTALNLEKDSDLE LTBP1S CynoNHTGRIKVVFTPSICKVTCTKGSCQNSCEKGNTTTLISENGHAADTLTATNFR  33VVLCHLPCMNGGQCSSRDKCQCPPNFTGKLCQIPVHGASVPKLYQHSQQPGKALGTHVIHSTHTLPLTVTSQQGVKVKFPPNIVNIHVKHPPEASVQIHQVSRIDGPTGQKTKEAQPGQSQVSYQGLPVQKTQTIHSTYSHQQVIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSKQEDCCGTVGTSWGFNKCQKCPKKPSYHGYNQMMECLPGYKRVNNTFCQDINECQLQGVCPNGECLNTMGSYRCTCKIGFGPDPTFSSCVPDPPVISEEKGPCYRLVSSGRQCMHPLSVHLTKQLCCCSVGKAWGPHCEKCPLPGTAAFKEICPGGMGYTVSGVHRRRPIHHHVGKGPVFVKPKNTQPVAKSTHPPPLPAKEEPVEALTFSREHGPGVAEPEVATAPPEKEIPSLDQEKTKLEPGQPQLSPGISTIHLHPQFPVVIEKTSPPVPVEVAPEASTSSASQVIAPTQVTEINECTVNPDICGAGHCINLPVRYTCICYEGYKFSEQQRKCVDIDECTQVQHLCSQGRCENTEGSFLCICPAGFMASEEGTNCIDVDECLRPDVCGEGHCVNTVGAFRCEYCDSGYRMTQRGRCEDIDECLNPSTCPDEQCVNSPGSYQCVPCTEGFRGWNGQCLDVDECLEPNVCTNGDCSNLEGSYMCSCHKGYTRTPDHKHCKDIDECQQGNLCVNGQCKNTEGSFRCTCGQGYQLSAAKDQCEDIDECQHHHLCAHGQCRNTEGSFQCVCDQGYRASGLGDHCEDINECLEDKSVCQRGDCINTAGSYDCTCPDGFQLDDNKTCQDINECEHPGLCGPQGECLNTEGSFHCVCQQGFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYQGFQAPQDGQGCVDVNECELLSGVCGEAFCENVEGSFLCVCADENQEYSPMTGQCRSRTSTDLDVEQPKEEKKECYYNLNDASLCDNVLAPNVTKQECCCTSGAGWGDNCEIFPCPVLGTAEFTEMCPKGKGFVPAGESSSEAGGENYKDADECLLFGQEICKNGFCLNTRPGYECYCKQGTYYDPVKLQCFDMDECQDPSSCIDGQCVNTEGSYNCFCTHPMVLDASEKRCIRPAESNEQIEETDVYQDLCWEHLSDEYVCSRPLVGKQTTYTECCCLYGEAWGMQCALCPMKDSDDYAQLCNIPVTGRRQPYGRDALVDFSEQYAPEADPYFIQDRFLNSFEELQAEECGILNGCENGRCVRVQEGYTCDCFDGYHLDTAKMTCVDVNECDELNNRMSLCKNAKCINTEGSYKCLCLPGYVPSDKPNYCTPLNTALNLEKDSDLE LTBP1S mouseNHTGRIKVVFTPSICKVTCTKGNCQNSCQKGNTTTLISENGHAADTLTATNFR  34VVICHLPCMNGGQCSSRDKCQCPPNFTGKLCQIPVLGASMPKLYQHAQQQGKALGSHVIHSTHTLPLTMTSQQGVKVKFPPNIVNIHVKHPPEASVQIHQVSRIDSPGGQKVKEAQPGQSQVSYQGLPVQKTQTVHSTYSHQQLIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSKQEDCCGTVGTSWGFNKCQKCPKKQSYHGYTQMMECLQGYKRVNNTFCQDINECQLQGVCPNGECLNTMGSYRCSCKMGFGPDPTFSSCVPDPPVISEEKGPCYRLVSPGRHCMHPLSVHLTKQICCCSVGKAWGPHCEKCPLPGTAAFKEICPGGMGYTVSGVHRRRPIHQHIGKEAVYVKPKNTQPVAKSTHPPPLPAKEEPVEALTSSWEHGPRGAEPEVVTAPPEKEIPSLDQEKTRLEPGQPQLSPGVSTIHLHPQFPVVVEKTSPPVPVEVAPEASTSSASQVIAPTQVTEINECTVNPDICGAGHCINLPVRYTCICYEGYKFSEQLRKCVDIDECAQVRHLCSQGRCENTEGSFLCVCPAGFMASEEGTNCIDVDECLRPDMCRDGRCINTAGAFRCEYCDSGYRMSRRGYCEDIDECLKPSTCPEEQCVNTPGSYQCVPCTEGFRGWNGQCLDVDECLQPKVCTNGSCTNLEGSYMCSCHRGYSPTPDHRHCQDIDECQQGNLCMNGQCRNTDGSFRCTCGQGYQLSAAKDQCEDIDECEHHHLCSHGQCRNTEGSFQCVCNQGYRASVLGDHCEDINECLEDSSVCQGGDCINTAGSYDCTCPDGFQLNDNKGCQDINECAQPGLCGSHGECLNTQGSFHCVCEQGFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYQGFQAPQDGQGCVDVNECELLSGVCGEAFCENVEGSFLCVCADENQEYSPMTGQCRSRVTEDSGVDRQPREEKKECYYNLNDASLCDNVLAPNVTKQECCCTSGAGWGDNCEIFPCPVQGTAEFTEMCPRGKGLVPAGESSYDTGGENYKDADECLLFGEEICKNGYCLNTQPGYECYCKQGTYYDPVKLQCFDMDECQDPNSCIDGQCVNTEGSYNCFCTHPMVLDASEKRCVQPTESNEQIEETDVYQDLCWEHLSEEYVCSRPLVGKQTTYTECCCLYGEAWGMQCALCPMKDSDDYAQLCNIPVTGRRRPYGRDALVDFSEQYGPETDPYFIQDRFLNSFEELQAEECGILNGCENGRCVRVQEGYTCDCFDGYHLDMAKMTCVDVNECSELNNRMSLCKNAKCINTEGSYKCLCLPGYIPSDKPNYCTPLNSALNLDKESDLE LTBP3S HumanETDECRLNQNICGHGECVPGPPDYSCHCNPGYRSHPQHRYCVDVNECEAEPCG 364PGRGICMNTGGSYNCHCNRGYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPPVCEDIDECRDPSSCPDGKCENKPGSFKCIACQPGYRSQGGGACRDVNECAEGSPCSPGWCENLPGSFRCTCAQGYAPAPDGRSCLDVDECEAGDVCDNGICSNTPGSFQCQCLSGYHLSRDRSHCEDIDECDFPAACIGGDCINTNGSYRCLCPQGHRLVGGRKCQDIDECSQDPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQHGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQRQCL LTBP3 HumanGPAGERGAGGGGALARERFKVVFAPVICKRTCLKGQCRDSCQQGSNMTLIGEN  35GHSTDTLTGSGFRVVVCPLPCMNGGQCSSRNQCLCPPDFTGRFCQVPAGGAGGGTGGSGPGLSRTGALSTGALPPLAPEGDSVASKHAIYAVQVIADPPGPGEGPPAQHAAFLVPLGPGQISAEVQAPPPVVNVRVHHPPEASVQVHRIESSNAESAAPSQHLLPHPKPSHPRPPTQKPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWGQSKCHKCPQLQYTGVQKPGPVRGEVGADCPQGYKRLNSTHCQDINECAMPGVCRHGDCLNNPGSYRCVCPPGHSLGPSRTQCIADKPEEKSLCFRLVSPEHQCQHPLTTRLTRQLCCCSVGKAWGARCQRCPTDGTAAFKEICPAGKGYHILTSHQTLTIQGESDFSLFLHPDGPPKPQQLPESPSQAPPPEDTEEERGVTTDSPVSEERSVQQSHPTATTTPARPYPELISRPSPPTMRWFLPDLPPSRSAVEIAPTQVTETDECRLNQNICGHGECVPGPPDYSCHCNPGYRSHPQHRYCVDVNECEAEPCGPGRGICMNTGGSYNCHCNRGYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPPVCEDIDECRDPSSCPDGKCENKPGSFKCIACQPGYRSQGGGACRDVNECAEGSPCSPGWCENLPGSFRCTCAQGYAPAPDGRSCLDVDECEAGDVCDNGICSNTPGSFQCQCLSGYHLSRDRSHCEDIDECDFPAACIGGDCINTNGSYRCLCPQGHRLVGGRKCQDIDECSQDPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQHGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQRQCLSPEEMDVDECQDPAACRPGRCVNLPGSYRCECRPPWVPGPSGRDCQLPESPAERAPERRDVCWSQRGEDGMCAGPLAGPALTFDDCCCRQGRGWGAQCRPCPPRGAGSHCPTSQSESNSFWDTSPLLLGKPPRDEDSSEEDSDECRCVSGRCVPRPGGAVCECPGGFQLDASRARCVDIDECRELNQRGLLCKSERCVNTSGSFRCVCKAGFARSRPHGACVPQRRR LTBP3 CYNOGPAGERGAGGGGALARERFKVVFAPVICKRTCLKGQCRDSCQQGSNMTLIGEN  36GHSTDTLTGSGFRVVVCPLPCMNGGQCSSRNQCLCPPDFTGRFCQVPAGGAGGGTGGSGPGLSRAGALSTGALPPLAPEGDSVASKHAIYAVQVIADPPGPGEGPPAQHAAFLVPLGPGQISAEVQAPPPVVNVRVHHPPEASVQVHRIESSNAEGAAPSQHLLPHPKPSHPRPPTQKPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWGQSKCHKCPQLQYTGVQKPGPVRGEVGADCPQGYKRLNSTHCQDINECAMPGVCRHGDCLNNPGSYRCVCPPGHSLGPSRTQCIADKPEEKSLCFRLVSPEHQCQHPLTTRLTRQLCCCSVGKAWGARCQRCPADGTAAFKEICPAGKGYHILTSHQTLTIQGESDFSLFLHPDGPPKPQQLPESPSQAPPPEDTEEERGVTTDSPVSEERSVQQSHPTATTSPARPYPELISRPSPPTMRWFLPDLPPSRSAVEIAPTQVTETDECRLNQNICGHGECVPGPPDYSCHCNPGYRSHPQHRYCVDVNECEAEPCGPGRGICMNTGGSYNCHCNRGYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPPVCEDIDECRDPSSCPDGKCENKPGSFKCIACQPGYRSQGGGACRDVNECAEGSPCSPGWCENLPGSFRCTCAQGYAPAPDGRSCVDVDECEAGDVCDNGICTNTPGSFQCQCLSGYHLSRDRSHCEDIDECDFPAACIGGDCINTNGSYRCLCPQGHRLVGGRKCQDIDECTQDPGLCLPHGACKNLQGSYVCVCDEGFTPTQDQHGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGAEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQRQCLSPEEMDVDECQDPAACRPGRCVNLPGSYRCECRPPWVPGPSGRDCQLPESPAERAPERRDVCWSQRGEDGMCAGPQAGPALTFDDCCCRQGRGWGAQCRPCPPRGAGSQCPTSQSESNSFWDTSPLLLGKPRRDEDSSEEDSDECRCVSGRCVPRPGGAVCECPGGFQLDASRARCVDIDECRELNQRGLLCKSERCVNTSGSFRCVCKAGFARSRPHGACVPQRRR LTBP3 MouseGPAGERGTGGGGALARERFKVVFAPVICKRTCLKGQCRDSCQQGSNMTLIGEN  37GHSTDTLTGSAFRVVVCPLPCMNGGQCSSRNQCLCPPDFTGRFCQVPAAGTGAGTGSSGPGLARTGAMSTGPLPPLAPEGESVASKHAIYAVQVIADPPGPGEGPPAQHAAFLVPLGPGQISAEVQAPPPVVNVRVHHPPEASVQVHRIEGPNAEGPASSQHLLPHPKPPHPRPPTQKPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWGQSKCHKCPQLQYTGVQKPVPVRGEVGADCPQGYKRLNSTHCQDINECAMPGNVCHGDCLNNPGSYRCVCPPGHSLGPLAAQCIADKPEEKSLCFRLVSTEHQCQHPLTTRLTRQLCCCSVGKAWGARCQRCPADGTAAFKEICPGKGYHILTSHQTLTIQGESDFSLFLHPDGPPKPQQLPESPSRAPPLEDTEEERGVTMDPPVSEERSVQQSHPTTTTSPPRPYPELISRPSPPTFHRFLPDLPPSRSAVEIAPTQVTETDECRLNQNICGHGQCVPGPSDYSCHCNAGYRSHPQHRYCVDVNECEAEPCGPGKGICMNTGGSYNCHCNRGYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPPICEDIDECRDPSTCPDGKCENKPGSFKCIACQPGYRSQGGGACRDVNECSEGTPCSPGWCENLPGSYRCTCAQYEPAQDGLSCIDVDECEAGKVCQDGICTNTPGSFQCQCLSGYHLSRDRSRCEDIDECDFPAACIGGDCINTNGSYRCLCPLGHRLVGGRKCKKDIDECSQDPGLCLPHACENLQGSYVCVCDEGFTLTQDQHGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLVPDGKRLHSGQQHCELCIPAHRDIDECILFGAEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQAQCLIPERWSTPQRDVKCAGASEERTACVWGPWAGPALTFDDCCCRQPRLGTQCRPCPPRGTGSQCPTSQSESNSFWDTSPLLLGKSPRDEDSSEEDSDECRCVSGRCVPRPGGAVCECPGGFQLDASRARCVDIDECRELNQRGLLCKSERCVNTSGSFRCVCKAGFTRSRPHGPACLSAAADDAAIAHTS VIDHRGYFH LTBP3SMouse ETDECRLNQNICGHGQCVPGPSDYSCHCNAGYRSHPQHRYCVDVNECEAEPCG 365PGKGICMNTGGSYNCHCNRGYRLHVGAGGRSCVDLNECTKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPPICEDIDECRDPSTCPDGKCENKPGSFKCIACQPGYRSQGGGACRDVNECSEGTPCSPGWCENLPGSYRCTCAQGYEPAQDGLSCIDVDECEAGKVCQDGICTNTPGSFQCQCLSGYHLSRDRSRCEDIDECDFPAACIGGDCINTNGSYRCLCPQGHRLVGGRKCQDIDECSQDPGLCLPHGACENLQGSYVCVCDEGFTLTQDQHGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECILFGAEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQRQCL

In an exemplary embodiment, inhibitors, e.g., antibodies, andantigen-binding portions thereof, that selectively bind LTBP1-TGFβ1and/or LTBP3-TGFβ1 do not bind to a complex containing TGFβ1 and GARP orLRRC33. In one embodiment, the antibodies, or antigen-binding portionsthereof, do not bind a GARP protein having a sequence set forth in SEQID NO:38 or SEQ ID NO:39, and do not bind to a complex containing saidGARP protein. In another embodiment, the inhibitors, e.g., antibodies,or antigen-binding portions thereof, do not bind a GARP protein having asequence set forth in SEQ ID NO:40 or SEQ ID NO:41, and do not bind to acomplex containing said GARP protein. In one embodiment, the inhibitors,e.g., antibodies, or antigen-binding portions thereof, do not bind aLRRC33 protein having a sequence set forth in SEQ ID NO:42 or SEQ IDNO:43, and do not bind a complex containing said LRRC33 protein. In oneembodiment, the inhibitors, e.g., antibodies, or antigen-bindingportions thereof, do not bind a GARP/LRRC33 chimera, e.g., theGARP/LRRC33 chimera set forth in SEQ ID NO:44.

TABLE 4 Exemplary GARP and LRRC33 amino acid sequences. Protein SequenceSEQ ID NO GARP AQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTETLDLSGNQLRSILASP 38LGFYTALRHLDLSTNEISFLQPGAFQALTHLEHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLQQLRVLDLSCNSIEAFQTASQPQAEFQLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSALPLSAPSGNASGRPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQGNALRDLPPYTFANLASLQRLNLQGNRVSPCGGPDEPGPSGCVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLSSNPGLEVATGALGGLEASLEVLALQGNGLMVLQVDLPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSFSLLPGSAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSHVRPEDCEKGGLKNINLIIILTFILVSAILLTTLAACCCVRRQKFNQQYKA sGARPAQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTETLDLSGNQLRSILASP 39LGFYTALRHLDLSTNEISFLQPGAFQALTHLEHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLQQLRVLDLSCNSIEAFQTASQPQAEFQLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSALPLSAPSGNASGRPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQGNALRDLPPYTFANLASLQRLNLQGNRVSPCGGPDEPGPSGCVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLSSNPGLEVATGALGGLEASLEVLALQGNGLMVLQVDLPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSFSLLPGSAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSHVRPEDCEKGGLKNIN GARPISQRREQVPCRTVNKEALCHGLGLLQVPSVLSLDIQALYLSGNQLQSILVSP 40 mouseLGFYTALRHLDLSDNQISFLQAGVFQALPYLEHLNLAHNRLATGMALNSGGLGRLPLLVSLDLSGNSLHGNLVERLLGETPRLRTLSLAENSLTRLARHTFWGMPAVEQLDLHSNVLMDIEDGAFEALPHLTHLNLSRNSLTCISDFSLQQLQVLDLSCNSIEAFQTAPEPQAQFQLAWLDLRENKLLHFPDLAVFPRLIYLNVSNNLIQLPAGLPRGSEDLHAPSEGWSASPLSNPSRNASTHPLSQLLNLDLSYNEIELVPASFLEHLTSLRFLNLSRNCLRSFEARQVDSLPCLVLLDLSHNVLEALELGTKVLGSLQTLLLQDNALQELPPYTFASLASLQRLNLQGNQVSPCGGPAEPGPPGCVDFSGIPTLHVLNMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATGALVGLEASLEVLELQGNGLTVLRVDLPCFLRLKRLNLAENQLSHLPAWTRAVSLEVLDLRNNSFSLLPGNAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFGSQEELSLSLVRPEDCEKGGLKNVNLILLLSFTLVSAIVLTTLATICFLRRQKLSQQYKA sGARPISQRREQVPCRTVNKEALCHGLGLLQVPSVLSLDIQALYLSGNQLQSILVS 41 mousePLGFYTALRHLDLSDNQISFLQAGVFQALPYLEHLNLAHNRLATGMALNSGGLGRLPLLVSLDLSGNSLHGNLVERLLGETPRLRTLSLAENSLTRLARHTFWGMPAVEQLDLHSNVLMDIEDGAFEALPHLTHLNLSRNSLTCISDFSLQQLQVLDLSCNSIEAFQTAPEPQAQFQLAWLDLRENKLLHFPDLAVFPRLIYLNVSNNLIQLPAGLPRGSEDLHAPSEGWSASPLSNPSRNASTHPLSQLLNLDLSYNEIELVPASFLEHLTSLRFLNLSRNCLRSFEARQVDSLPCLVLLDLSHNVLEALELGTKVLGSLQTLLLQDNALQELPPYTFASLASLQRLNLQGNQVSPCGGPAEPGPPGCVDFSGIPTLHVLNMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATGALVGLEASLEVLELQGNGLTVLRVDLPCFLRLKRLNLAENQLSHLPAWTRAVSLEVLDLRNNSFSLLPGNAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFGSQEELSLSLVRPEDCEKGGLKNVN LRRC33 (alsoMELLPLWLCLGFHFLTVGWRNRSGTATAASQGVCKLVGGAADCRGQSLASV 42 known as NRROS;PSSLPPHARMLTLDANPLKTLWNHSLQPYPLLESLSLHSCHLERISRGAFQ Uniprot AccessionEQGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLSGNALTEDMAALML No. Q86YC3)QNLSSLRSVSLAGNTIMRLDDSVFEGLERLRELDLQRNYIFEIEGGAFDGLAELRHLNLAFNNLPCIVDFGLTRLRVLNVSYNVLEWFLATGGEAAFELETLDLSHNQLLFFPLLPQYSKLRTLLLRDNNMGFYRDLYNTSSPREMVAQFLLVDGNVTNITTVSLWEEFSSSDLADLRFLDMSQNQFQYLPDGFLRKMPSLSHLNLHQNCLMTLHIREHEPPGALTELDLSHNQLSELHLAPGLASCLGSLRLFNLSSNQLLGVPPGLFANARNITTLDMSHNQISLCPLPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQGTSLTYLDLSSNWGVLNGSLAPLQDVAPMLQVLSLRNMGLHSSFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGLRTIYLSQNPYDCCGVDGWGALQHGQTVADWAMVTCNLSSKIIRVTELPGGVPRDCKWERLDLGLLYLVLILPSCLTLLVACTVIVLTFKKPLLQVIKSRCHWSSVY*Native signal peptide is depicted in bold font. soluble LRRC33MDMRVPAQLLGLLLLWFSGVLGWRNRSGTATAASQGVCKLVGGAADCRGQS 43 (sLRRC33)LASVPSSLPPHARMLTLDANPLKTLWNHSLQPYPLLESLSLHSCHLERISRGAFQEQGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLSGNALTEDMAALMLQNLSSLRSVSLAGNTIMRLDDSVFEGLERLRELDLQRNYIFEIEGGAFDGLAELRHLNLAFNNLPCIVDFGLTRLRVLNVSYNVLEWFLATGGEAAFELETLDLSHNQLLFFPLLPQYSKLRTLLLRDNNMGFYRDLYNTSSPREMVAQFLLVDGNVTNITTVSLWEEFSSSDLADLRFLDMSQNQFQYLPDGFLRKMPSLSHLNLHQNCLMTLHIREHEPPGALTELDLSHNQLSELHLAPGLASCLGSLRLFNLSSNQLLGVPPGLFANARNITTLDMSHNQISLCPLPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQGTSLTYLDLSSNWGVLNGSLAPLQDVAPMLQVLSLRNMGLHSSFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGLRTIYLSQNPYDCCGVDGWGALQHGQTVADWAMVTCNLSSKIIRVTELPGGVPRDCKWERLDLGLHHHHHH*Modified human kappa light chain signal peptide isdepicted in bold font. **Histidine tag is underlined. Human LRRC33-MDMRVPAQLLGLLLLWFSGVLG WRNRSGTATAASQGVCKLVGGAADCRGQS 44LASVPSSLPPHARMLTLDANPLKTLWNHSLQPYPLLESLSLHSCHLERISR GARP chimeraGAFQEQGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLSGNALTEDMAALMLQNLSSLRSVSLAGNTIMRLDDSVFEGLERLRELDLQRNYIFEIEGGAFDGLAELRHLNLAFNNLPCIVDFGLTRLRVLNVSYNVLEWFLATGGEAAFELETLDLSHNQLLFFPLLPQYSKLRTLLLRDNNMGFYRDLYNTSSPREMVAQFLLVDGNVTNITTVSLWEEFSSSDLADLRFLDMQSNQFQYLPDGFLRKMPSLSHLNLHQNCLMTLHIREHEPPGALTELDLSHNQLSELHLAPGLASCLGSLRLFNLSSNQLLGVPPGLFANARNITTLDMSHNQISLCPLPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQGTSLTYLDLSSNWGVLNGSLAPLQDVAPMLQVLSLRNMGLHSSFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGLRTIYLSQNPYDCCGVDGWGALQHGQTVADWAMVTCNLSSKIIRVTELPGGVPRDCKWERLDLGL LIIILTFILVSAILLTTLAACCCVRRQKFNQQYKA*Modified human kappa light chain signal peptide isdepicted in bold font. **LRRC33 ectodomain is underlined.# GARP transmembrane domain is italicized.## GARP intracellular tail is double underlined.

In another aspect, the invention provides methods of inhibiting TGFβ1activation in the context of LTBP1 and/or LTBP3. In one embodiment, themethod comprises exposing a LTBP1-proTGFβ1 complex or a LTBP3-proTGFβ1complex an inhibitor, an antibody or antigen-binding portion thereof,and/or a pharmaceutical composition described herein. For example, inone embodiment, the inhibitor is an inhibitor of extracellularmatrix-associated TGFβ1 activation, which selectively binds aLTBP1/3-presented proTGFβ1 latent complex. In one embodiment, theinhibitor does not inhibit immune cell-associated TGFβ1 activation, forexample, immune cell-associated TGFβ1 activation that results fromactivation of a GARP-presented proTGFβ1 latent complex. In anotherembodiment, the antibody, or antigen-binding portion thereof,selectively binds an LTBP1-proTGFβ1 latent complex and/or anLTBP3-proTGFβ1 latent complex, thereby modulating release of matureTGFβ1 growth factor from the latent complex, wherein the antibody, orantigen-binding portion thereof, does not bind mature TGFβ1 alone or aGARP-proTGFβ1 latent complex. In one embodiment, the antibody, orantigen-binding portion thereof, inhibits the release of mature TGFβ1from the LTBP1-proTGFβ1 complex and/or the LTBP3-proTGFβ1 complex. Inone embodiment, the antibody, or antigen-binding portion thereof, doesnot inhibit the release of mature TGFβ1 from a GARP-proTGFβ1 complex ora LRRC33-proTGFβ1 complex.

In one embodiment, the method is performed in vitro. In anotherembodiment, the method is performed in vivo. In one embodiment, theLTBP1-proTGFβ1 complex or the LTBP3-proTGFβ1 complex is in anextracellular matrix. The extracellular matrix can comprise, forexample, fibrillin and/or fibronectin. In some embodiments, theextracellular matrix comprises a protein comprising an RGD motif.

In some embodiments of the foregoing aspects, the antibody, orantigen-binding portion thereof, does not stimulate immune effectorcells. In one embodiment, the antibody, or antigen-binding portionthereof, inhibits the release of mature TGFβ1 from a LTBP1-proTGFβ1complex and/or a LTBP3-proTGFβ1 complex, and does not inhibit therelease of mature TGFβ1 from a GARP-proTGFβ1 complex and/or anLRRC33-proTGFβ1 complex.

In some embodiments, inhibitors, e.g., antibodies, of the presentdisclosure that selectively bind to a LTBP1-TGFβ1 complex and/or aLTBP3-TGFβ1 complex can bind the complex with relatively high affinity,e.g., with a dissociation constant (K_(D)) less than 10⁻⁶M, 10⁻⁷ M,10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M or lower. In one embodiment, anantibody, or antigen-binding portion thereof, binds a LTBP1-TGFβ1complex and/or a LTBP3-TGFβ1 complex with a dissociation constant(K_(D)) of about 10⁻⁸M, about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹ M,about 10⁻¹² M, or about 10⁻¹³ M. For example, antibodies thatselectively bind to a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complexcan bind the complex with an affinity between 5 pM and 500 nM, e.g.,between 10 pM and 100 nM, e.g., between 50 pM and 50 nM. In oneembodiment, the antibody, or antigen-binding fragment thereof, can binda LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with an affinity ofless than about 300 nm, for example about 20 nM or lower, about 10 nM orlower, about 500 pM or lower, or about 5 pM or lower. For example, theantibody, or antigen-binding fragment thereof, can bind a LTBP1-TGFβ1complex and/or a LTBP3-TGFβ1 complex with an affinity of about 1 nm toabout 350 nm, from about 10 nm to about 200 nm, from about 15 nm toabout 250 nm, from about 20 nm to about 200 nm, about 1 nm, about 20 nm,about 25 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm,about 250 nm, about 300 nm, or about 500 pm.

The disclosure also includes antibodies or antigen-binding fragmentsthat compete with any of the antibodies described herein for binding toa LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex. In some embodiments,such antibodies have an affinity for the complex of 50 nM or lower(e.g., 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower,or 5 pM or lower). The affinity and binding kinetics of antibodies (orantigen-binding fragments thereof) that selectively bind to aLTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex can be tested using anysuitable method, including but not limited to biosensor technology(e.g., OCTET or BIACORE).

In one embodiment, the antibodies, or antigen-binding fragments thereof,of the present disclosure do not compete with antibody SR-Abl forbinding to a human LTBP1-proTGFβ1 complex.

Aspects of the disclosure relate to antibodies that compete orcross-compete with any of the antibodies provided herein. The term“compete”, as used herein with regard to an antibody, means that a firstantibody binds to an epitope (e.g., an epitope of a LTBP1-TGFβ1 complexand/or an epitope of a LTBP3-TGFβ1 complex) in a manner sufficientlysimilar to the binding of a second antibody, such that the result ofbinding of the first antibody with its epitope is detectably decreasedin the presence of the second antibody compared to the binding of thefirst antibody in the absence of the second antibody. The alternative,where the binding of the second antibody to its epitope is alsodetectably decreased in the presence of the first antibody, can, butneed not be the case. That is, a first antibody can inhibit the bindingof a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its epitope or ligand, whether to the same, greater,or lesser extent, the antibodies are said to “cross-compete” with eachother for binding of their respective epitope(s). Both competing andcross-competing antibodies are within the scope of this disclosure.Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof), the skilled artisanwould appreciate that such competing and/or cross-competing antibodiesare encompassed and can be useful for the methods and/or compositionsprovided herein.

Aspects of the disclosure relate to antibodies that compete orcross-compete with any of the specific antibodies, or antigen-bindingportions thereof, as provided herein, e.g., an antibody having one ormore CDR sequences (1, 2, 3, 4, 5, or 6 CDR sequences) set forth inTable 5. In one embodiment, the invention provides antibodies, andantigen-binding fragments thereof, that compete or cross-compete with anantibody having heavy chain CDR sequences comprising SEQ ID NO:1, SEQ IDNO:2, and SEQ ID NO:3 as set forth in Table 5, and/or light chain CDRsequences comprising SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 as setforth in Table 5. In one embodiment, the invention provides antibodiesthat compete or cross-compete with an antibody, or antigen-bindingportion thereof, having a heavy chain variable region sequencecomprising SEQ ID NO:7, and/or a light chain variable region sequencecomprising SEQ ID NO:8. In some embodiments, an antibody, orantigen-binding portion thereof, binds at or near the same epitope asany of the antibodies provided herein. In some embodiments, an antibody,or antigen-binding portion thereof, binds near an epitope if it bindswithin 15 or fewer amino acid residues of the epitope. In someembodiments, any of the antibody, or antigen-binding portion thereof, asprovided herein, binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15 amino acid residues of an epitope that is bound by any of theantibodies provided herein.

In another embodiment, provided herein is an antibody, orantigen-binding portion thereof, competes or cross-competes for bindingto any of the antigens provided herein (e.g., a LTBP1-TGFβ1 complexand/or a LTBP3-TGFβ1 complex) with an equilibrium dissociation constant,K_(D), between the antibody and the protein of less than 10⁻⁶ M. Inother embodiments, an antibody competes or cross-competes for binding toany of the antigens provided herein with a K_(D) in a range from 10 ¹¹ Mto 10⁻⁶ M. In other embodiments, an antibody competes or cross-competesfor binding to a human LTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1complex with a K_(D) of <50 nM as determined by a suitable in vitrobinding assay, e.g., BLI, such as Octet®. In other embodiments, anantibody competes or cross-competes for binding to a human LTBP1-TGFβ1complex and/or a human LTBP3-TGFβ1 complex with a K_(D) of <10 nM asdetermined by a suitable in vitro binding assay, e.g., BLI, such asOctet.

In some embodiments, the antibody or antigen-binding portion competes orcross-competes with an antibody having a heavy chain variable regionsequence and light chain variable region sequence of Ab42, as set forthin Table 6 (e.g., SEQ ID NOs: 318 and 319, respectively). The antibodymay compete or cross-compete for binding to a human LTBP1-TGFβ1 complexand/or a human LTBP3-TGFβ1 complex with a K_(D) of <10 nM as determinedby a suitable in vitro binding assay, e.g., BLI, such as Octet. Theantibody may compete or cross-compete for binding to a human LTBP1-TGFβ1complex and/or a human LTBP3-TGFβ1 complex with a K_(D) of <5 nM asdetermined by a suitable in vitro binding assay, e.g., BLI, such asOctet. The antibody may bind to a human LTBP1-TGFβ1 complex and a humanLTBP3-TGFβ1 complex with a K_(D) of <5 nM as determined by a suitable invitro binding assay, e.g., BLI, such as Octet. The antibody may not showany detectable binding to a human GARP-proTGFβ1 complex in a suitable invitro binding assay, such as BLI (e.g., Octet). The antibody may notshow detectable binding to a human GARP-proTGFβ1 complex, as measured byBLI, under the same assay conditions as used to measure binding to humanLTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1 complex. Alternatively,or in addition, the antibody or antigen-binding portion may bind (e.g.,selectively bind) a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-TGFβ1 complex with a K_(D) that is at least 50 times lower thanthe K_(D) when binding to a human GARP-proTGFβ1 complex (and optionallyat least 50 times lower than the K_(D) when binding to a humanLRRC33-proTGFβ1 complex) under the same assay conditions.

In some embodiments, the antibody competes or cross-competes with anantibody having a heavy chain variable region sequence and light chainvariable region sequence of Ab63, as set forth in Table 6 (e.g., SEQ IDNOs: 360 and 361, respectively). The antibody may compete orcross-compete for binding to a human LTBP1-TGFβ1 complex and/or a humanLTBP3-TGFβ1 complex with a K_(D) of <10 nM as determined by a suitablein vitro binding assay, e.g., BLI, such as Octet. The antibody maycompete or cross-compete for binding to a human LTBP1-TGFβ1 complexand/or a human LTBP3-TGFβ1 complex with a K_(D) of <5 nM as determinedby a suitable in vitro binding assay, e.g., BLI, such as Octet. Theantibody may bind to a human LTBP1-TGFβ1 complex and a human LTBP3-TGFβ1complex with a K_(D) of <5 nM as determined by a suitable in vitrobinding assay, e.g., BLI, such as Octet. The antibody may not show anydetectable binding to a human GARP-proTGFβ1 complex in a suitable invitro binding assay, such as BLI (e.g., Octet). The antibody may notshow detectable binding to a human GARP-proTGFβ1 complex, as measured byBLI, under the same assay conditions as used to measure binding to humanLTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1 complex. Alternatively,or in addition, the antibody or antigen-binding portion may bind (e.g.,selectively bind) a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-TGFβ1 complex with a K_(D) that is at least 50 times lower thanthe K_(D) when binding to a human GARP-proTGFβ1 complex (and optionallyat least 50 times lower than the K_(D) when binding to a humanLRRC33-proTGFβ1 complex) under the same assay conditions.

In further embodiments, the antibody which selectively binds a humanLTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex may not showmeaningful binding (e.g., may not show a response of more than 0.1 units(nm)) on exposure to a human GARP-proTGFβ1 complex in a BLI assay (e.g.,Octet) when the human GARP-proTGFβ1 complex is at a concentration of 200nM.

In some embodiments, provided herein is an anti-TGFβ1 antibody, orantigen-binding portion thereof, that competes for binding with anantibody, or antigen-binding portion thereof, described herein. In someembodiments, provided herein is an anti-TGFβ1 antibody, orantigen-binding portion thereof, that binds to the same epitope as anantibody, or antigen-binding portion thereof, described herein.

The antibodies provided herein can be characterized using any suitablemethods. For example, one method is to identify the epitope to which theantigen binds, or “epitope mapping.” There are many suitable methods formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. In an additionalexample, epitope mapping can be used to determine the sequence to whichan antibody binds. The epitope can be a linear epitope, i.e., containedin a single stretch of amino acids, or a conformational epitope formedby a three-dimensional interaction of amino acids that may notnecessarily be contained in a single stretch (primary structure linearsequence). In some embodiments, the epitope is a TGFβ1 epitope that isonly available for binding by the antibody, or antigen-binding portionthereof, described herein, when the TGFβ1 is in a LTBP1-TGFβ1 complexand/or a LTBP3-TGFβ1 complex. In some embodiments, the epitope ispresent on a LTBP1/3-TGFβ1 complex, and is not present on a GARP-TGFβ1complex and/or a LRRC33-TGFβ1 complex. In some embodiments, the epitopeis available due to a conformational change in LTBP1/3 and/or TGFβ1 thatoccurs when LTBP1/3 and TGFβ1 form a complex. In this embodiment, theepitope is not present in LTBP1/3 or TGFβ1 when the proteins are notassociated in a complex. In one embodiment, the epitope is present onTGFβ1, when TGFβ1 is in a complex with LTBP1 or LTBP3. In anotherembodiment, the epitope is present on LTBP1, when LTBP1 is in a complexwith TGFβ1. In another embodiment, the epitope is present on LTBP3, whenLTBP3 is in a complex with TGFβ1. In another embodiment, the epitopecomprises residues from both LTBP1 and TGFβ1. In another embodiment, theepitope comprises residues from both LTBP3 and TGFβ1. Peptides ofvarying lengths (e.g., at least 4-6 amino acids long) can be isolated orsynthesized (e.g., recombinantly) and used for binding assays with anantibody. In another example, the epitope to which the antibody bindscan be determined in a systematic screen by using overlapping peptidesderived from the target antigen sequence and determining binding by theantibody. According to the gene fragment expression assays, the openreading frame encoding the target antigen is fragmented either randomlyor by specific genetic constructions and the reactivity of the expressedfragments of the antigen with the antibody to be tested is determined.The gene fragments may, for example, be produced by PCR and thentranscribed and translated into protein in vitro, in the presence ofradioactive amino acids. The binding of the antibody to theradioactively labeled antigen fragments is then determined byimmunoprecipitation and gel electrophoresis. Certain epitopes can alsobe identified by using large libraries of random peptide sequencesdisplayed on the surface of phage particles (phage libraries).Alternatively, a defined library of overlapping peptide fragments can betested for binding to the test antibody in simple binding assays. In anadditional example, mutagenesis of an antigen-binding domain, domainswapping experiments and alanine scanning mutagenesis can be performedto identify residues required, sufficient, and/or necessary for epitopebinding. For example, domain swapping experiments can be performed usinga mutant of a target antigen in which various fragments of theLTBP1-TGFβ1 complex or LTBP3-TGFβ1 complex have been replaced (swapped)with sequences from a closely related, but antigenically distinctprotein, such as another member of the TGFβ protein family (e.g.,GDF11). By assessing binding of the antibody to the mutant of theLTBP1-TGFβ1 complex and/or LTBP3-TGFβ1 complex, the importance of theparticular antigen fragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using otherantibodies known to bind to the same antigen to determine whether anantibody binds to the same epitope as the other antibodies. Competitionassays are well known to those of skill in the art.

Further, the interaction of the any of the antibodies provided hereinwith one or more residues in a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1complex can be determined by routine technology. For example, a crystalstructure can be determined, and the distances between the residues in aLTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, and one or moreresidues in the antibody, can be determined accordingly. Based on suchdistance, whether a specific residue in a LTBP1/3-TGFβ1 complexinteracts with one or more residues in the antibody can be determined.Further, suitable methods, such as competition assays and targetmutagenesis assays, can be applied to determine the preferential bindingof a candidate antibody.

In some embodiments, the antibodies, or antigen-binding portionsthereof, of the present invention that selectively bind to a LTBP1-TGFβ1complex and/or a LTBP3-TGFβ1 complex include one or more ofcomplementary determining regions (CDRs) shown in Table 5. In someembodiments, the invention provides a nucleic acid molecule that encodesan antibody, or antigen-binding portion thereof, that selectively bindsto a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, as describedherein. In one embodiment, the nucleic acid molecules encode one or moreof the CDR sequences shown in Table 5.

TABLE 5 Complementary determining regions of the heavychain (CDRHs) and the light chain (CDRLs) of  SR-AB2, SR-AB10, SR-AB13, SR-AB22, SR-AB23, SR-AB31,SR-AB34, SR-AB37, and SR-AB38 to SR-AB64 asdetermined using the Kabat numbering scheme. Antibody SR-AB2 CDRH1GYTFTSYG (SEQ ID NO: 1) CDRH2 ISAYNGNT (SEQ ID NO: 2) CDRH3 ARAPLGNFDS(SEQ ID NO: 3) CDRL1 SGSIASNY (SEQ ID NO: 4) CDRL2 EDN (SEQ ID NO: 5)CDRL3 QSYDSSNHPVV (SEQ ID NO: 6) Antibody SR-AB10 CDRH1 FTFNNYPIH(SEQ ID NO: 94) CDRH2 VMSYDGINKYYADSVKG (SEQ ID NO: 95) CDRH3ARPRIAARRGGFDY (SEQ ID NO: 96) CDRL1 TRSSGNIDNNYVQ (SEQ ID NO: 97) CDRL2EDNQRPS (SEQ ID NO: 98) CDRL3 QSYDSDNQGVV (SEQ ID NO: 99) AntibodySR-AB13 CDRH1 GSISSSSYYWG (SEQ ID NO: 100) CDRH2 SISYSGSTYY(SEQ ID NO: 101) CDRH3 ARDPSYDSIAGMDV (SEQ ID NO: 102) CDRL1 RASQSISSYLN(SEQ ID NO: 103) CDRL2 AASNLQS (SEQ ID NO: 104) CDRL3 QQSFDFPFT(SEQ ID NO: 105) Antibody SR-AB22 CDRH1 FTFRSYVMH (SEQ ID NO: 108) CDRH2VISHEGSLKYYADSVKG (SEQ ID NO: 109) CDRH3 AVPRIAARRGGFGY (SEQ ID NO: 110)CDRL1 TRSSGNIDNNYVQ (SEQ ID NO: 111) CDRL2 EDNQRPS (SEQ ID NO: 112)CDRL3 QSYDSDNQGVV (SEQ ID NO: 113) Antibody SR-AB23 CDRH1 FTFRSYVMH(SEQ ID NO: 116) CDRH2 VISHEGSLKYYADSVKG (SEQ ID NO: 117) CDRH3ARPRIAARRGGFGY (SEQ ID NO: 118) CDRL1 TRSSGNIDNNYVQ (SEQ ID NO: 119)CDRL2 EDNQRPS (SEQ ID NO: 120) CDRL3 QSYDSDNQGVV (SEQ ID NO: 121)Antibody SR-AB31 CDRH1 FTFRSYVMH (SEQ ID NO: 124) CDRH2VISHEGSLKYYADSVKG (SEQ ID NO: 125) CDRH3 AVPRIAARRGGFGY (SEQ ID NO: 126)CDRL1 TRSSGNIDNNYVQ (SEQ ID NO: 127) CDRL2 EDNQRPS (SEQ ID NO: 128)CDRL3 QSYDFNNQGVV (SEQ ID NO: 129) Antibody SR-AB34 CDRH1 FTFRSYVMH(SEQ ID NO: 130) CDRH2 VISHEGSLKYYADSVKG (SEQ ID NO: 131) CDRH3AVPRIAARRGGFGY (SEQ ID NO: 132) CDRL1 TRSSGNIDNNYVQ (SEQ ID NO: 133)CDRL2 EDNQRPS (SEQ ID NO: 134) CDRL3 QSYDYDAQGVV (SEQ ID NO: 135)Antibody SR-AB37 CDRH1 FTFRSYVMH (SEQ ID NO: 136) CDRH2VISHEGSLKYYADSVKG (SEQ ID NO: 137) CDRH3 AVPRIAARRGGFGY (SEQ ID NO: 138)CDRL1 TRSSGLIDDNYVQ (SEQ ID NO: 139) CDRL2 EDNQRPS (SEQ ID NO: 140)CDRL3 QSYDSDLQRVV (SEQ ID NO: 141) Antibody SR-AB38 CDRH1 FTFRSYVMH(SEQ ID NO: 142) CDRH2 VISHEGSLKYYADSVKG (SEQ ID NO: 143) CDRH3AVPRIAARRGGFGY (SEQ ID NO: 144) CDRL1 TRSSGSIDNNYVQ (SEQ ID NO: 145)CDRL2 EDFIRPS (SEQ ID NO: 146) CDRL3 QSYDDDLQGVV (SEQ ID NO: 147)Antibody SR-AB39 CDRH1 FTFRSYVMH (SEQ ID NO: 148) CDRH2VISHEGSLKYYADSVKG (SEQ ID NO: 149) CDRH3 AVPRIAARRGGFGY (SEQ ID NO: 150)CDRL1 TRSSGLIDDNYVQ (SEQ ID NO: 151) CDRL2 EDAQRPS (SEQ ID NO: 152)CDRL3 QSYDHDEQGVV (SEQ ID NO: 153) Antibody SR-AB40 CDRH1 FTFRSYVMH(SEQ ID NO: 154) CDRH2 VISHEGSLKYYADSVKG (SEQ ID NO: 155) CDRH3ARPRIAARRGGFGY (SEQ ID NO: 156) CDRL1 TRSSGNIDNNYVQ (SEQ ID NO: 157)CDRL2 EDNQRPS (SEQ ID NO: 158) CDRL3 QSYDYSNQGVV (SEQ ID NO: 159)Antibody SR-AB41 CDRH1 FTFRSYVMH (SEQ ID NO: 160) CDRH2VISHEGSLKYYADSVKG (SEQ ID NO: 161) CDRH3 ARPRIAARRGGFGY (SEQ ID NO: 162)CDRL1 TRSSGNIDNNYVQ (SEQ ID NO: 163) CDRL2 EDNQRPS (SEQ ID NO: 164)CDRL3 QSYDYDNQAVV (SEQ ID NO: 165) Antibody SR-AB42 CDRH1 FTFRSYVMH(SEQ ID NO: 166) CDRH2 VISHEGSLKYYADSVKG (SEQ ID NO: 167) CDRH3ARPRIAARRGGFGY (SEQ ID NO: 168) CDRL1 TRSSGNIDNNYVQ (SEQ ID NO: 169)CDRL2 EDNQRPS (SEQ ID NO: 170) CDRL3 QSYDYDTQGVV (SEQ ID NO: 171)Antibody SR-AB43 CDRH1 FTFRSYVMH (SEQ ID NO: 172) CDRH2VISHEGSLKYYADSVKG (SEQ ID NO: 173) CDRH3 ARPRIAARRGGFGY (SEQ ID NO: 174)CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 175) CDRL2 EDNVRPS (SEQ ID NO: 176)CDRL3 QSYDSDNQRVV (SEQ ID NO: 177) Antibody SR-AB44 CDRH1 FTFRSYVMH(SEQ ID NO: 178) CDRH2 VISHEGSLKYYADSVKG (SEQ ID NO: 179) CDRH3ARPRIAARRGGFGY (SEQ ID NO: 180) CDRL1 TRSHGNIDDNYVQ (SEQ ID NO: 181)CDRL2 EDNVRPS (SEQ ID NO: 182) CDRL3 QSYDSDNQLVV (SEQ ID NO: 183)Antibody SR-AB45 CDRH1 FTFRSYVMH (SEQ ID NO: 184) CDRH2VISHEGSLKYYADSVKG (SEQ ID NO: 185) CDRH3 ARPRIAARRGGFGY (SEQ ID NO: 186)CDRL1 TRSSGAIDDNYVQ (SEQ ID NO: 187) CDRL2 EDFQRPS (SEQ ID NO: 188)CDRL3 QSYDDDLQGVV (SEQ ID NO: 189) Antibody SR-AB46 CDRH1 FTFRSYVMH(SEQ ID NO: 190) CDRH2 VISHEGSGKYYADSVKG (SEQ ID NO: 191) CDRH3ARPRIAARRGGFGS (SEQ ID NO: 192) CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 193)CDRL2 EDNVRPS (SEQ ID NO: 194) CDRL3 QSYDSDNQRVV (SEQ ID NO: 195)Antibody SR-AB47 CDRH1 FTFRSYVMH (SEQ ID NO: 196) CDRH2VISHEGSGKYYADSVKG (SEQ ID NO: 197) CDRH3 ARPRIAARRGGFGS (SEQ ID NO: 198)CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 199) CDRL2 EDNVRPS (SEQ ID NO: 200)CDRL3 QSYDYDNQAVV (SEQ ID NO: 201) Antibody SR-AB48 CDRH1 FTFRSYVMH(SEQ ID NO: 202) CDRH2 VISHEGSGKYYADSVKG (SEQ ID NO: 203) CDRH3ARPRIAARRGGFGS (SEQ ID NO: 204) CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 205)CDRL2 EDNVRPS (SEQ ID NO: 206) CDRL3 QSYDYDTQGVV (SEQ ID NO: 207)Antibody SR-AB49 CDRH1 FTFRSYVMH (SEQ ID NO: 208) CDRH2VISHEGSGKYYADSVKG (SEQ ID NO: 209) CDRH3 ARPRIAARRGGFGS (SEQ ID NO: 210)CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 211) CDRL2 EDNVRPS (SEQ ID NO: 212)CDRL3 QGYDWDTQGVV (SEQ ID NO: 213) Antibody SR-AB50 CDRH1 FTFRSYVMH(SEQ ID NO: 214) CDRH2 VISHEGSGKYYADSVKG (SEQ ID NO: 215) CDRH3ARPRIAARRGGFGT (SEQ ID NO: 216) CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 217)CDRL2 EDNVRPS (SEQ ID NO: 218) CDRL3 QSYDSDNQRVV (SEQ ID NO: 219)Antibody SR-AB51 CDRH1 FTFRSYVMH (SEQ ID NO: 220) CDRH2VISHEGSGKYYADSVKG (SEQ ID NO: 221) CDRH3 ARPRIAARRGGFGT (SEQ ID NO: 222)CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 223) CDRL2 EDNVRPS (SEQ ID NO: 224)CDRL3 QSYDYDNQAVV (SEQ ID NO: 225) Antibody SR-AB52 CDRH1 FTFRSYVMH(SEQ ID NO: 226) CDRH2 VISHEGSGKYYADSVKG (SEQ ID NO: 227) CDRH3ARPRIAARRGGFGT (SEQ ID NO: 228) CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 229)CDRL2 EDNVRPS (SEQ ID NO: 230) CDRL3 QSYDYDTQGVV (SEQ ID NO: 231)Antibody SR-AB53 CDRH1 FTFRSYVMH (SEQ ID NO: 232) CDRH2VISHEGSGKYYADSVKG (SEQ ID NO: 233) CDRH3 ARPRIAARRGGFGT (SEQ ID NO: 234)CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 235) CDRL2 EDNVRPS (SEQ ID NO: 236)CDRL3 QGYDWDTQGVV (SEQ ID NO: 237) Antibody SR-AB54 CDRH1 FTFRSYVMH(SEQ ID NO: 238) CDRH2 VISHEGSGKYYADSVKG (SEQ ID NO: 239) CDRH3ALPRIAARRGGFGS (SEQ ID NO: 240) CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 241)CDRL2 EDNVRPS (SEQ ID NO: 242) CDRL3 QSYDSDNQRVV (SEQ ID NO: 243)Antibody SR-AB55 CDRH1 FTFRSYVMH (SEQ ID NO: 244) CDRH2VISHEGSGKYYADSVKG (SEQ ID NO: 245) CDRH3 ALPRIAARRGGFGS (SEQ ID NO: 246)CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 247) CDRL2 EDNVRPS (SEQ ID NO: 248)CDRL3 QSYDYDNQAVV (SEQ ID NO: 249) Antibody SR-AB56 CDRH1 FTFRSYVMH(SEQ ID NO: 250) CDRH2 VISHEGSGKYYADSVKG (SEQ ID NO: 251) CDRH3ALPRIAARRGGFGS (SEQ ID NO: 252) CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 253)CDRL2 EDNVRPS (SEQ ID NO: 254) CDRL3 QSYDYDTQGVV (SEQ ID NO: 255)Antibody SR-AB57 CDRH1 FTFRSYVMH (SEQ ID NO: 256) CDRH2VISHEGSGKYYADSVKG (SEQ ID NO: 257) CDRH3 ALPRIAARRGGFGS (SEQ ID NO: 258)CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 259) CDRL2 EDNVRPS (SEQ ID NO: 260)CDRL3 QGYDWDTQGVV (SEQ ID NO: 261) Antibody SR-AB58 CDRH1 FTFRSYVMH(SEQ ID NO: 262) CDRH2 VISHEGSGKYYADSVKG (SEQ ID NO: 263) CDRH3ALPRIAARRGGFGT (SEQ ID NO: 264) CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 265)CDRL2 EDNVRPS (SEQ ID NO: 266) CDRL3 QSYDSDNQRVV (SEQ ID NO: 267)Antibody SR-AB59 CDRH1 FTFRSYVMH (SEQ ID NO: 268) CDRH2VISHEGSGKYYADSVKG (SEQ ID NO: 269) CDRH3 ALPRIAARRGGFGT (SEQ ID NO: 270)CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 271) CDRL2 EDNVRPS (SEQ ID NO: 272)CDRL3 QSYDYDNQAVV (SEQ ID NO: 273) Antibody SR-AB60 CDRH1 FTFRSYVMH(SEQ ID NO: 274) CDRH2 VISHEGSGKYYADSVKG (SEQ ID NO: 275) CDRH3ALPRIAARRGGFGT (SEQ ID NO: 276) CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 277)CDRL2 EDNVRPS (SEQ ID NO: 278) CDRL3 QSYDYDTQGVV (SEQ ID NO: 279)Antibody SR-AB61 CDRH1 FTFRSYVMH (SEQ ID NO: 280) CDRH2VISHEGSGKYYADSVKG (SEQ ID NO: 281) CDRH3 ALPRIAARRGGFGT (SEQ ID NO: 282)CDRL1 TRSSGNIDYNYVQ (SEQ ID NO: 283) CDRL2 EDNVRPS (SEQ ID NO: 284)CDRL3 QGYDWDTQGVV (SEQ ID NO: 285) Antibody SR-AB62 CDRH1 GSIRSSSYYWG(SEQ ID NO: 286) CDRH2 SISYSATTYY (SEQ ID NO: 287) CDRH3 ASDPSYDSAAGMDV(SEQ ID NO: 288) CDRL1 RASKVISSYLN (SEQ ID NO: 289) CDRL2 YASSLQS(SEQ ID NO: 290) CDRL3 QQSNDWPFT (SEQ ID NO: 291) Antibody SR-AB63 CDRH1GSIRSSSYYWG (SEQ ID NO: 292) CDRH2 SISYSATTYY (SEQ ID NO: 293) CDRH3AGDPSYDSIAGMQV (SEQ ID NO: 294) CDRL1 RASQSISSYLN (SEQ ID NO: 295) CDRL2AASNLQS (SEQ ID NO: 296) CDRL3 QQSFDWPLT (SEQ ID NO: 297) AntibodySR-AB64 CDRH1 GSIRSSSYYWG (SEQ ID NO: 298) CDRH2 SISYSATTYY(SEQ ID NO: 299) CDRH3 AGDPSYDSIAGMQV (SEQ ID NO: 300) CDRL1 RASQSISYYLN(SEQ ID NO: 301) CDRL2 SASSRQS (SEQ ID NO: 302) CDRL3 QQGFDFPLT(SEQ ID NO: 303)

In some embodiments, antibodies of the present invention thatselectively bind to a LTBP1-TGFβ3 complex and/or a LTBP3-TGFβ complexinclude any antibody, or antigen-binding portion thereof, comprising aCDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, asprovided in Table 5. In some embodiments, antibodies that selectivelybind to a LTBP1-TGFβ3 complex and/or a LTBP3-TGFβ complex include CDRH1,CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 as provided in Table 5.

The present invention also provides a nucleic acid sequence that encodesa molecule comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, orcombinations thereof, as provided in Table 5.

Antibody heavy and light chain CDR3 domains may play a particularlyimportant role in the binding specificity/affinity of an antibody for anantigen. Accordingly, in some embodiments, the antibodies, orantigen-binding portions thereof, that selectively bind to a LTBP1-TGFβcomplex and/or a LTBP3-TGFβ complex, or the nucleic acid molecules thatencode these antibodies, or antigen-binding portions thereof, caninclude at least the heavy and/or light chain CDR3 of the antibody shownin Table 5.

Aspects of the invention relate to a monoclonal antibody, orantigen-binding portion thereof, that binds selectively to a LTBP1-TGFβcomplex and/or a LTBP3-TGFβ complex, and that comprises sixcomplementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, and CDRL3. The antibody, or antigen-binding portion thereof mayhave the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of one of theantibodies (e.g., Ab42) shown in Table 5.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ IDNO: 1. In some embodiments, CDRH2 comprises a sequence as set forth inSEQ ID NO: 2. In some embodiments, CDRH3 comprises a sequence as setforth in SEQ ID NO: 3. In some embodiments, CDRL1 comprises a sequenceas set forth in SEQ ID NO: 4. In some embodiments, CDRL2 comprises asequence as set forth in SEQ ID NO: 5. In some embodiments, CDRL3comprises a sequence as set forth in SEQ ID NO: 6.

In one aspect, the invention provides an isolated antibody, or anantigen-binding fragment thereof , that specifically binds a humanLTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex and does notbind a human GARP-proTGFβ1 complex; wherein the antibody or theantigen-binding fragment thereof does not bind mature TGFβ1, matureTGFβ2 or mature TGFβ3; wherein the antibody or the antigen-bindingfragment thereof is a fully human or humanized antibody orantigen-binding fragment thereof, wherein the antibody or theantigen-binding fragment thereof comprises at least three CDRs selectedfrom the following, optionally comprising up to one or more amino acidchanges for each of the CDRs: CDR-H1: SEQ ID NO:1; CDR-H2: SEQ ID NO:2;CDR-H3: SEQ ID NO:3; CDR-L1: SEQ ID NO:4; CDR-L2: SEQ ID NO:5; and,CDR-L3: SEQ ID NO:6. In some embodiments, the one or more amino acidchanges comprises up to 1, 2, 3, 4, 5, or 6 amino acid changes for eachof the CDRs.

In some embodiments (e.g., as for antibody SR-AB2, shown in Table 5),the antibody, or antigen-binding portion thereof, that selectively bindsto a LTBP1-TGFβ complex and/or a LTBP3-TGFβ3 complex comprises: a CDRH1comprising an amino acid sequence as set forth in SEQ ID NO: 1, a CDRH2comprising an amino acid sequence as set forth in SEQ ID NO: 2, a CDRH3comprising an amino acid sequence as set forth in SEQ ID NO: 3, a CDRL1comprising an amino acid sequence as set forth in SEQ ID NO: 4, a CDRL2comprising an amino acid sequence as set forth in SEQ ID NO: 5, and aCDRL3 comprising an amino acid sequence as set forth in SEQ ID NO: 6.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:3 and a light chain variable region comprising a CDR3 having the aminoacid sequence of SEQ ID NO: 6. In some embodiments, the antibody, orantigen-binding portion thereof, comprises a heavy chain variable regioncomprising a complementarity determining region 2 (CDR2) having theamino acid sequence of SEQ ID NO: 2 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 5. Insome embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:1 and a light chain variable region comprising a CDR1 having the aminoacid sequence of SEQ ID NO: 4.

The amino acid sequences of the heavy chain variable region (HCVR) andthe light chain variable region (LCVR) of the antibody set forth inTable 5 (e.g., SR-AB2) are provided in Table 6.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ IDNO: 94. In some embodiments, CDRH2 comprises a sequence as set forth inSEQ ID NO: 95. In some embodiments, CDRH3 comprises a sequence as setforth in SEQ ID NO: 96. In some embodiments, CDRL1 comprises a sequenceas set forth in SEQ ID NO: 97. In some embodiments, CDRL2 comprises asequence as set forth in SEQ ID NO: 98. In some embodiments, CDRL3comprises a sequence as set forth in SEQ ID NO: 99.

In one aspect, the invention provides an isolated antibody, or anantigen-binding fragment thereof , that specifically binds a humanLTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex and does notbind a human GARP-proTGFβ1 complex; wherein the antibody or theantigen-binding fragment thereof does not bind mature TGFβ1, matureTGFβ2 or mature TGFβ3; wherein the antibody or the antigen-bindingfragment thereof is a fully human or humanized antibody orantigen-binding fragment thereof, wherein the antibody or theantigen-binding fragment thereof comprises at least three CDRs selectedfrom the following, optionally comprising one or more amino acid changesfor each of the CDRs: CDR-H1: SEQ ID NO:94; CDR-H2: SEQ ID NO:95;CDR-H3: SEQ ID NO:96; CDR-L1: SEQ ID NO:97; CDR-L2: SEQ ID NO:98; and,CDR-L3: SEQ ID NO:99. In some embodiments, the one or more amino acidchanges comprises up to 1, 2, 3, 4, 5, or 6 amino acid changes for eachof the CDRs.

In some embodiments (e.g., as for antibody SR-AB10, shown in Table 5),the antibody, or antigen-binding portion thereof, that selectively bindsto a LTBP1-TGFβ complex and/or a LTBP3-TGFβ3 complex comprises: a CDRH1comprising an amino acid sequence as set forth in SEQ ID NO: 94, a CDRH2comprising an amino acid sequence as set forth in SEQ ID NO: 95, a CDRH3comprising an amino acid sequence as set forth in SEQ ID NO: 96, a CDRL1comprising an amino acid sequence as set forth in SEQ ID NO: 97, a CDRL2comprising an amino acid sequence as set forth in SEQ ID NO: 98, and aCDRL3 comprising an amino acid sequence as set forth in SEQ ID NO: 99.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:96 and a light chain variable region comprising a CDR3 having the aminoacid sequence of SEQ ID NO: 99. In some embodiments, the antibody, orantigen-binding portion thereof, comprises a heavy chain variable regioncomprising a complementarity determining region 2 (CDR2) having theamino acid sequence of SEQ ID NO: 95 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 98. Insome embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:94 and a light chain variable region comprising a CDR1 having the aminoacid sequence of SEQ ID NO: 97.

The amino acid sequences of the heavy chain variable region (HCVR) andthe light chain variable region (LCVR) of the antibody set forth inTable 5 (e.g., SR-AB10) are provided in Table 6.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ IDNO: 100. In some embodiments, CDRH2 comprises a sequence as set forth inSEQ ID NO: 101. In some embodiments, CDRH3 comprises a sequence as setforth in SEQ ID NO: 102. In some embodiments, CDRL1 comprises a sequenceas set forth in SEQ ID NO: 103. In some embodiments, CDRL2 comprises asequence as set forth in SEQ ID NO: 104. In some embodiments, CDRL3comprises a sequence as set forth in SEQ ID NO: 105.

In one aspect, the invention provides an isolated antibody, or anantigen-binding fragment thereof , that specifically binds a humanLTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex and does notbind a human GARP-proTGFβ1 complex; wherein the antibody or theantigen-binding fragment thereof does not bind mature TGFβ1, matureTGFβ2 or mature TGFβ3; wherein the antibody or the antigen-bindingfragment thereof is a fully human or humanized antibody orantigen-binding fragment thereof, wherein the antibody or theantigen-binding fragment thereof comprises at least three CDRs selectedfrom the following, optionally comprising one or more amino acid changesfor each of the CDRs: CDR-H1: SEQ ID NO:100; CDR-H2: SEQ ID NO:101;CDR-H3: SEQ ID NO:102; CDR-L1: SEQ ID NO:103; CDR-L2: SEQ ID NO:104;and, CDR-L3: SEQ ID NO:105. In some embodiments, the one or more aminoacid changes comprises up to 1, 2, 3, 4, 5, or 6 amino acid changes foreach of the CDRs.

In some embodiments (e.g., as for antibody SR-AB13, shown in Table 5),the antibody, or antigen-binding portion thereof, that selectively bindsto a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex comprises: a CDRH1comprising an amino acid sequence as set forth in SEQ ID NO: 100, aCDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 101,a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO:102, a CDRL1 comprising an amino acid sequence as set forth in SEQ IDNO: 103, a CDRL2 comprising an amino acid sequence as set forth in SEQID NO: 104, and a CDRL3 comprising an amino acid sequence as set forthin SEQ ID NO: 105.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:102 and a light chain variable region comprising a CDR3 having the aminoacid sequence of SEQ ID NO: 105. In some embodiments, the antibody, orantigen-binding portion thereof, comprises a heavy chain variable regioncomprising a complementarity determining region 2 (CDR2) having theamino acid sequence of SEQ ID NO: 101 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 104. Insome embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:100 and a light chain variable region comprising a CDR1 having the aminoacid sequence of SEQ ID NO: 103.

The amino acid sequences of the heavy chain variable region (HCVR) andthe light chain variable region (LCVR) of the antibody set forth inTable 5 (e.g., SR-AB13) are provided in Table 6.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ IDNO: 124. In some embodiments, CDRH2 comprises a sequence as set forth inSEQ ID NO: 125. In some embodiments, CDRH3 comprises a sequence as setforth in SEQ ID NO: 126. In some embodiments, CDRL1 comprises a sequenceas set forth in SEQ ID NO: 127. In some embodiments, CDRL2 comprises asequence as set forth in SEQ ID NO: 128. In some embodiments, CDRL3comprises a sequence as set forth in SEQ ID NO: 129.

In one aspect, the invention provides an isolated antibody, or anantigen-binding fragment thereof, that specifically binds a humanLTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex; wherein theantibody or the antigen-binding fragment thereof does not bind matureTGFβ1, mature TGFβ2 or mature TGFβ3; wherein the antibody or theantigen-binding fragment thereof is a fully human or humanized antibodyor antigen-binding fragment thereof, wherein the antibody or theantigen-binding fragment thereof comprises at least three (optionallyall six) CDRs selected from the following, optionally comprising up toone or more amino acid changes for each of the CDRs: CDR-H1: SEQ IDNO:124; CDR-H2: SEQ ID NO:125; CDR-H3: SEQ ID NO:126; CDR-Ll: SEQ IDNO:127; CDR-L2: SEQ ID NO:128; and, CDR-L3: SEQ ID NO:129. In someembodiments, the one or more amino acid changes comprises up to 1, 2, 3,4, 5, or 6 amino acid changes for each of the CDRs.

In some embodiments (e.g., as for antibody SR-AB31, shown in Table 5),the antibody, or antigen-binding portion thereof, that selectively bindsto a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex comprises: a CDRH1comprising an amino acid sequence as set forth in SEQ ID NO: 124, aCDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 125,a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO:126, a CDRL1 comprising an amino acid sequence as set forth in SEQ IDNO: 127, a CDRL2 comprising an amino acid sequence as set forth in SEQID NO: 128, and a CDRL3 comprising an amino acid sequence as set forthin SEQ ID NO: 129.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:126 and a light chain variable region comprising a CDR3 having the aminoacid sequence of SEQ ID NO: 129. In some embodiments, the antibody, orantigen-binding portion thereof, comprises a heavy chain variable regioncomprising a complementarity determining region 2 (CDR2) having theamino acid sequence of SEQ ID NO: 125 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 128. Insome embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:124 and a light chain variable region comprising a CDR1 having the aminoacid sequence of SEQ ID NO: 127.

The amino acid sequences of the HCVR and the LCVR of the antibody setforth in Table 5 (e.g., SR-AB31) are provided in Table 6.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ IDNO: 166. In some embodiments, CDRH2 comprises a sequence as set forth inSEQ ID NO: 167. In some embodiments, CDRH3 comprises a sequence as setforth in SEQ ID NO: 168. In some embodiments, CDRL1 comprises a sequenceas set forth in SEQ ID NO: 169. In some embodiments, CDRL2 comprises asequence as set forth in SEQ ID NO: 170. In some embodiments, CDRL3comprises a sequence as set forth in SEQ ID NO: 171.

In one aspect, the invention provides an isolated antibody, or anantigen-binding fragment thereof, that specifically binds a humanLTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex; wherein theantibody or the antigen-binding fragment thereof does not bind matureTGFβ1, mature TGFβ2 or mature TGFβ3; wherein the antibody or theantigen-binding fragment thereof is a fully human or humanized antibodyor antigen-binding fragment thereof, wherein the antibody or theantigen-binding fragment thereof comprises at least three (optionallyall six) CDRs selected from the following, optionally comprising up toone or more amino acid changes for each of the CDRs: CDR-H1: SEQ IDNO:166; CDR-H2: SEQ ID NO:167; CDR-H3: SEQ ID NO:168; CDR-Ll: SEQ IDNO:169; CDR-L2: SEQ ID NO:170; and, CDR-L3: SEQ ID NO:171. In someembodiments, the one or more amino acid changes comprises up to 1, 2, 3,4, 5, or 6 amino acid changes for each of the CDRs.

In some embodiments (e.g., as for antibody SR-AB42, shown in Table 5),the antibody, or antigen-binding portion thereof, that selectively bindsto a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex comprises: a CDRH1comprising an amino acid sequence as set forth in SEQ ID NO: 166, aCDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 167,a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO:168, a CDRL1 comprising an amino acid sequence as set forth in SEQ IDNO: 169, a CDRL2 comprising an amino acid sequence as set forth in SEQID NO: 170, and a CDRL3 comprising an amino acid sequence as set forthin SEQ ID NO: 171.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:168 and a light chain variable region comprising a CDR3 having the aminoacid sequence of SEQ ID NO: 171. In some embodiments, the antibody, orantigen-binding portion thereof, comprises a heavy chain variable regioncomprising a complementarity determining region 2 (CDR2) having theamino acid sequence of SEQ ID NO: 167 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 170. Insome embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:166 and a light chain variable region comprising a CDR1 having the aminoacid sequence of SEQ ID NO: 169.

The amino acid sequences of the HCVR and the LCVR of the antibody setforth in Table 5 (e.g., SR-AB42) are provided in Table 6.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ IDNO: 292. In some embodiments, CDRH2 comprises a sequence as set forth inSEQ ID NO: 293. In some embodiments, CDRH3 comprises a sequence as setforth in SEQ ID NO: 294. In some embodiments, CDRL1 comprises a sequenceas set forth in SEQ ID NO: 295. In some embodiments, CDRL2 comprises asequence as set forth in SEQ ID NO: 296. In some embodiments, CDRL3comprises a sequence as set forth in SEQ ID NO: 297.

In one aspect, the invention provides an isolated antibody, or anantigen-binding fragment thereof, that specifically binds a humanLTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex; wherein theantibody or the antigen-binding fragment thereof does not bind matureTGFβ1, mature TGFβ2 or mature TGFβ3; wherein the antibody or theantigen-binding fragment thereof is a fully human or humanized antibodyor antigen-binding fragment thereof, wherein the antibody or theantigen-binding fragment thereof comprises at least three (optionallyall six) CDRs selected from the following, optionally comprising up toone or more amino acid changes for each of the CDRs: CDR-H1: SEQ IDNO:292; CDR-H2: SEQ ID NO:293; CDR-H3: SEQ ID NO:294; CDR-L1: SEQ IDNO:295; CDR-L2: SEQ ID NO:296; and, CDR-L3: SEQ ID NO:297. In someembodiments, the one or more amino acid changes comprises up to 1, 2, 3,4, 5, or 6 amino acid changes for each of the CDRs.

In some embodiments (e.g., as for antibody SR-AB63, shown in Table 5),the antibody, or antigen-binding portion thereof, that selectively bindsto a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex comprises: a CDRH1comprising an amino acid sequence as set forth in SEQ ID NO: 292, aCDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 293,a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO:294, a CDRL1 comprising an amino acid sequence as set forth in SEQ IDNO: 295, a CDRL2 comprising an amino acid sequence as set forth in SEQID NO: 296, and a CDRL3 comprising an amino acid sequence as set forthin SEQ ID NO: 297.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:294 and a light chain variable region comprising a CDR3 having the aminoacid sequence of SEQ ID NO: 297. In some embodiments, the antibody, orantigen-binding portion thereof, comprises a heavy chain variable regioncomprising a complementarity determining region 2 (CDR2) having theamino acid sequence of SEQ ID NO: 293 and a light chain variable regioncomprising a CDR2 having the amino acid sequence of SEQ ID NO: 296. Insome embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable region comprising a complementaritydetermining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:292 and a light chain variable region comprising a CDR1 having the aminoacid sequence of SEQ ID NO: 295.

The amino acid sequences of the HCVR and the LCVR of the antibody setforth in Table 5 (e.g., SR-AB63) are provided in Table 6.

Ten additional antibodies (Ab3-Ab12) were developed that specificallybind to a LTBP1-TGFβ3 complex and/or a LTBP3-TGFβ complex, and inhibitrelease of mature TGFβ presented in the context of LTBP1/3. Table 6 alsoprovides the HCVR and LCVR amino acid sequences of these additional LTBPcontext-specific antibodies, in addition to the HCVR and LCVR amino acidsequences of the antibodies referred to in Table 5.

TABLE 6Heavy Chain Variable Region Sequence and Light Chain Variable Region Sequence ofAntibodies that Specifically Bind a LTBP1/3-TGFβ1 Complex AntibodyHCVR Sequence LCVR Sequence SR-AB2 QVQLVQSGAEVKKPGASVKVSCKANFMLTQPHSVSESPGKTVTISCTRSS SGYTFTSYGISWVRQAPGQGLEWMGSIASNYVQWYQQRPGSSPTTVIYE GWISAYNGNTNYAQKLQGRVTMTTDNQRPSGVPDRFSGSIDSSSNSASLTI DTSTSTAYMELRSLRSDDTAVYYCASGLKTEDEADYYCQSYDSSNHPVVF RAPLGNFDSWGQGTMVTVSS (SEQGGGTKLTVL (SEQ ID NO: 8) ID NO: 7) SR-AB3 QMQLVQSGAEVKKPGASVKVSCKAQSGLTQPASVSGSPGQSVTISCTGTS SGYTFTSYGISWVRQAPGQGLEWMSDVGGYNYASWYQQHPGKAPKLMI GWISAYNGNTNYAQKLQGRVTMTTYDVSKRPSGVPDRFSGSKSGNTASL NTSTSTAYMELRSLRSDDTAVYYCATISGLQAEDEADYYCSSYTSSSTYVF RDDYYYYGMDVWGQGTLVTVSSGTGTKLTVL (SEQ ID NO: 75) (SEQ ID NO: 74) SR-AB4QVQLQQWGAGLLKPSETLSLTCAV QSELTQSPSASGTPGQRVTISCSGSNYGGSFSGYYWSWIRQPPGKGLEWI SNIGTNTVNWYQQFPGTAPKLLIYYGEIIHSGSTNYNPSLKSRVTISVDTSK NDQRPSGVSDRFSGSRSGTSASLAINNQFSLKLSSVTAADTAVYYCARGV GLQSEDEADYYCATWDDSLSGVVFGLGRFDPWGQGTLVTVSS (SEQ ID GGGTKLTVL (SEQ ID NO: 77) NO: 76) SR-AB5QVQLQQWGAGLLKPSETLSLTCAV QSELTQSPSASGTPGQRVTISCSGSNYGGSFSGYYWSWIRQPPGKGLEWI SNIGTNTVNWYQQFPGTAPKLLIYYGEINHSGSTNYNPSLKSRVTISVDTS NDQRPSGVSDRFSGSRSGTSASLAINKNQFSLKLSSVTAADTAVYYCARG GLQSEDEADYYCATWDDSLSGVVFVGLGRFDPWGQGTLVTVSS (SEQ GGGTKLTVL (SEQ ID NO: 79) ID NO: 78) SR-AB6QVQLQQSGPGLVRPSQTLSLTCAISG NFMLTQPHSVSESPGKTVTISCTRSSDSVSSNGAAWNWIRQSPSRGLEWL GSIASNYVQWYQQRPGSAPTTVIYDGRTYYRSKWYNDYAVSVKSRITINP DKQRPSGIPDRFSGSIDSSSNSASLTIDTSKNQFSLKLTSVTPEDTAVYYCA SGLKTEDEADYYCQSYDSSNVVFGRGEDWGYAFDIWGQGTLVTVSS GGTKVTVL (SEQ ID NO: 81) (SEQ ID NO: 80) SR-AB7QVQLVQSGAEVKKPGASVKVSCKA QSELTQAPSVSVAPGQTARITCGGNSGYTFTSYGISWVRQAPGQGLEWM NIGGRSKSVHWYQHKLGQAPVLIVGWISAYDGNTNYAQKLQGRVTMTT YDNTDRPSGISERFSGSSSVNAATLTDTSTSTAYMELSSLRSDDTAVYYCA ITTAEAGDEGDYYCQVWDVSTDHV RNPYYYYMDVWGQGTTVTVSSVFGGGTKVTVL (SEQ ID NO: 83) (SEQ ID NO: 82) SR-AB8QVQLVESGAEVKKPGASVKVSCKA NFMLTQPHSVSESPGKTVTISCTGSSSGYTFTGYYMHWVRQAPGQGLEW GSIASNYVQWYQQRPGSSPTTVIYEMGWINPNGGGTNYAQKFQGRVTM DNQRPSGVPDRFSGSIDSSSNSASLTITRDTSISTAYMELSRLRSDDTAVYY SGLKTEDEADYYCQSYDDNYHVIFCANRRRGSAFDIWGQGTLVTVSS GGGTKLTVL (SEQ ID NO: 85) (SEQ ID NO: 84) SR-AB9QVQLVESGGALVQPGGSLRLSCAAS NFMLTQPHSVSESPGRTLTIPCFRSSGFTFSSYAMHWVRQAPGKGLEWV GNIGDSYVHWYQQRPGSAPTTVIYRAVISYDGSNKYYADSVKGRFTISRD DSQRPSGVPDRFSGSIDFSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEAAYYCQSYDRSNQWVF KETGYGFGLFWGQGTMVTVSSGGGTKLTVL (SEQ ID NO: 87) (SEQ ID NO: 86) SR-AB10QLQLQESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFNNYPIHWVRQAPGKGLEWVA GNIDNNYVQWYQQRPGSSPTTVIYEVMSYDGINKYYADSVKGRFTISRDN DNQRPSGVPDRFSGSIDSSSNSASLTISKNTLYLQMNSLRAEDTAVYYCAR SGLKTEDEADYYCQSYDSDNQGVVPRIAARRGGFDYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 89) (SEQ ID NO: 88)SR-AB11 QVQLVQSGAEVKKPGASVKVSCKA NFMLTQPHSVSESPGKTVTISCTRSSSGYTFTSYGISWVRQAPGQGLEWM GSIASNYVQWYQQRPGSAPTTVIYEGWISAYNGNTDYAQKLQGRVTMTT DNQRPSGVPDRFSGSIDSSSNSASLTIDTSTSTAYMELRGLRSDDTAVYYC SGLKTEDEADYYCQSYDSSNHVVFARAPLGNFDSWGQGTLVTVSS (SEQ GGGTKVTVL (SEQ ID NO: 91) ID NO: 90) SR-AB12EVQLLESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFPNYAMSWVRQAPGKGLEWVS GSIASNYVQWYQQRPGSSPTTVIYEAISGSGGSTYYADSVKGRFTISRDNS DNQRPSGVPDRFSGSIDSSSNSASLTIKNTLYLQMNSLRAEDTAVYYCAKD SGLKTEDEADYYCQSYDSSIVVFGGLEGGYYWDYYYYGMDVWGQGTL GTQLTVL (SEQ ID NO: 93) VTVSS (SEQ ID NO: 92)SR-AB13 QLQLQESGPGLVKPSETLSLTCTVSG DIQLTQSPSSLSASVGDRVTITCRASGSISSSSYYWGWIRQPPGKGLEWIG QSISSYLNWYQQKPGKAPKLLIYAASISYSGSTYYNPSLKSRVTISVDTSK SNLQSGVPSRFSGSGSGTDFTLTISSLNQFSLKLSSVTAADTAVYYCARDPS QPEDFATYYCQQSFDFPFTFGGGTKYDSIAGMDVWGQGTTVTVSS (SEQ VEIK (SEQ ID NO: 107) ID NO: 106) SR-AB22QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDNNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DNQRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDSDNQGVVVPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 115) (SEQ ID NO: 114)SR-AB23 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDNNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DNQRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDSDNQGVVRPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 123) (SEQ ID NO: 122)SR-AB31 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDNNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DNQRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDFNNQGVVVPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 304) (SEQ ID NO: 305)SR-AB34 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDNNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DNQRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDAQGVVVPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 306) (SEQ ID NO: 307)SR-AB37 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GLIDDNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DNQRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDSDLQRVVVPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 308) (SEQ ID NO: 309)SR-AB38 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GSIDNNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DFIRPSGVPDRFSGSIDSSSNSASLTISNSKNTLYLQMNSLRAEDTAVYYCA GLKTEDEADYYCQSYDDDLQGVVFVPRIAARRGGFGYWGQGTLVTVSS GGGTKLTVL (SEQ ID NO: 310) (SEQ ID NO: 311)SR-AB39 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GLIDDNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DAQRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDHDEQGVVVPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 312) (SEQ ID NO: 313)SR-AB40 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDNNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DNQRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYSNQGVVRPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 314) (SEQ ID NO: 315)SR-AB41 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDNNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DNQRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDNQAVVRPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 316) (SEQ ID NO: 317)SR-AB42 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDNNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DNQRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDTQGVVRPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 318) (SEQ ID NO: 319)SR-AB43 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDSDNQRVVRPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 320) (SEQ ID NO: 321)SR-AB44 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSHGFTFRSYVMHWVRQAPGKGLEWV GNIDDNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDSDNQLVVRPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 322) (SEQ ID NO: 323)SR-AB45 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GAIDDNYVQWYQQRPGSSPTTVIYEAVISHEGSLKYYADSVKGRFTISRD DFQRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDDDLQGVVRPRIAARRGGFGYWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 324) (SEQ ID NO: 325)SR-AB46 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDSDNQRVVRPRIAARRGGFGSWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 326) (SEQ ID NO: 327)SR-AB47 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDNQAVVRPRIAARRGGFGSWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 328) (SEQ ID NO: 329)SR-AB48 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDTQGVVRPRIAARRGGFGSWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 330) (SEQ ID NO: 331)SR-AB49 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQGYDWDTQGVRPRIAARRGGFGSWGQGTLVTVSS VFGGGTKLTVL (SEQ ID NO: 332) (SEQ ID NO: 333)SR-AB50 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDSDNQRVVRPRIAARRGGFGTWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 334) (SEQ ID NO: 335)SR-AB51 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDNQAVVRPRIAARRGGFGTWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 336) (SEQ ID NO: 337)SR-AB52 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDTQGVVRPRIAARRGGFGTWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 338) (SEQ ID NO: 339)SR-AB53 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQGYDWDTQGVRPRIAARRGGFGTWGQGTLVTVSS VFGGGTKLTVL (SEQ ID NO: 340) (SEQ ID NO: 341)SR-AB54 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDSDNQRVVLPRIAARRGGFGSWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 342) (SEQ ID NO: 343)SR-AB55 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDNQAVVLPRIAARRGGFGSWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 344) (SEQ ID NO: 345)SR-AB56 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDTQGVVLPRIAARRGGFGSWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 346) (SEQ ID NO: 347)SR-AB57 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQGYDWDTQGVLPRIAARRGGFGSWGQGTLVTVSS VFGGGTKLTVL (SEQ ID NO: 348) (SEQ ID NO: 349)SR-AB58 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDSDNQRVVLPRIAARRGGFGTWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 350) (SEQ ID NO: 351)SR-AB59 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDNQAVVLPRIAARRGGFGTWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 352) (SEQ ID NO: 353)SR-AB60 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQSYDYDTQGVVLPRIAARRGGFGTWGQGTLVTVSS FGGGTKLTVL (SEQ ID NO: 354) (SEQ ID NO: 355)SR-AB61 QVQLVESGGGVVQPGRSLRLSCAAS NFMLTQPHSVSESPGKTVTISCTRSSGFTFRSYVMHWVRQAPGKGLEWV GNIDYNYVQWYQQRPGSSPTTVIYEAVISHEGSGKYYADSVKGRFTISRD DNVRPSGVPDRFSGSIDSSSNSASLTINSKNTLYLQMNSLRAEDTAVYYCA SGLKTEDEADYYCQGYDWDTQGVLPRIAARRGGFGTWGQGTLVTVSS VFGGGTKLTVL (SEQ ID NO: 356) (SEQ ID NO: 357)SR-AB62 QLQLQESGPGLAKPSETLSLTCTVSG DIQMTQSPSSLSASVGDRVTITCRASGSIRSSSYYWGWIRQPPGKGLEWIG KVISSYLNWYQQKPGKAPKLLIYYASISYSATTYYNPSLKSRVTISVDTSK SSLQSGVPSRFSGSGSGTDFTLTISSLNQFSLKLSSVTAADTAVYYCASDPS QPEDFATYYCQQSNDWPFTFGGGT YDSAAGMDVWGQGTTVTVSSKVEIK (SEQ ID NO: 358) (SEQ ID NO: 359) SR-AB63QLQLQESGPGLVKPSETLSLTCTVSG DIQLTQSPSSLSASVGDRVTITCRASGSIRSSSYYWGWIRQPPGKGLEWIG QSISSYLNWYQQKPGKAPKLLIYAASISYSATTYYNPSLKSRVTISVDTSK SNLQSGVPSRFSGSGSGTDFTLTISSLNQFSLKLSSVTAADTAVYYCAGDPS QPEDFATYYCQQSFDWPLTFGGGT YDSIAGMQVWGQGTTVTVSSKVEIK (SEQ ID NO: 360) (SEQ ID NO: 361) SR-AB64QLQLQESGPGLVKPSETLSLTCTVSG DIQMTQSPSSLSASVGDRVTITCRASGSIRSSSYYWGWIRQPPGKGLEWIG QSISYYLNWYQQKPGKAPKLLIYSASISYSATTYYNPSLKSRVTISVDTSK SSRQSGVPSRFSGSGSGTDFTLTISSLNQFSLKLSSVTAADTAVYYCAGDPS QPEDFATYYCQQGFDFPLTFGGGTK YDSIAGMQVWGQGTTVTVSSVEIK (SEQ ID NO: 362) (SEQ ID NO: 363)

Aspects of the invention relate to a monoclonal antibody, orantigen-binding portion thereof, that binds selectively to a LTBP1-TGFβcomplex and/or a LTBP3-TGFβ complex, and that comprises a heavy chainvariable region sequence and a light chain variable region sequence.

In one aspect, the invention provides an isolated antibody or anantigen-binding fragment thereof, that specifically binds a humanLTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex and does notbind a human GARP-proTGFβ1 complex; wherein the antibody or theantigen-binding fragment thereof does not bind mature TGFβ1, matureTGFβ2 or mature TGFβ3; wherein the antibody or the antigen-bindingfragment thereof is a fully human or humanized antibody or anantigen-binding fragment thereof; wherein the antibody or theantigen-binding fragment thereof comprises a variable heavy chain havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to any one of the variable region aminoacid sequences set forth in Table 6.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 7, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO:88, SEQ ID NO: 90, SEQ ID NO: 92, or SEQ ID NO: 106. In one embodiment,the antibody, or antigen-binding fragment thereof, comprises a lightchain variable region having an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ IDNO: 8, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91,SEQ ID NO: 93, or SEQ ID NO: 107.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 7 and/or a light chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 8. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 8. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 7 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 74 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 75. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 74 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 75. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 74 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 75.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 76 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 77. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 76 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 77. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 76 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 77.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 78 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 79. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 78 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 79. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 78 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 79.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 80 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 81. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 80 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 81. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 80 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 81.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 82 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 83. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 82 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 83. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 82 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 83.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 84 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 85. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 84 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 85. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 84 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 85.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 86 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 87. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 86 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 87. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 86 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 87.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 88 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 89. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 88 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 89. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 88 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 89.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 90 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 91. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 90 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 91. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 90 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 91.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 92 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 93. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 92 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 93. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 92 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 93.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 106 and/or a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 107. In one embodiment, theantibody, or antigen-binding fragment thereof, comprises a heavy chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 106 ora light chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 107. In one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 106 and a light chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 107.

In one aspect, the invention provides an isolated antibody or anantigen-binding fragment thereof, that specifically binds a humanLTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex. The antibodymay selectively bind a human LTBP1-proTGFβ complex and/or a humanLTBP3-proTGFβ complex. The antibody or the antigen-binding fragmentthereof may not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3. Theantibody or the antigen-binding fragment thereof may be a fully human orhumanized antibody or an antigen-binding fragment thereof. The antibodyor the antigen-binding fragment thereof may comprises a variable heavychain having an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to any one of the variableregion amino acid sequences set forth in Table 6. In some embodiments,the level of identity is at least 95% (optionally at least 98%).

Accordingly, in one embodiment, the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 318 and/or a light chainvariable region having an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 319.The antibody, or antigen-binding fragment thereof, may comprise a heavychain variable region having an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ IDNO: 318 or a light chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 319. The antibody, or antigen-binding fragmentthereof, may comprise a heavy chain variable region having an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to SEQ ID NO: 318 and a light chain variable region havingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 319.

In another embodiment, the antibody, or antigen-binding fragmentthereof, comprises a heavy chain variable region having an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to SEQ ID NO: 360 and/or a light chain variable regionhaving an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 361. The antibody, orantigen-binding fragment thereof, may comprise a heavy chain variableregion having an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 360 or alight chain variable region having an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO: 361. The antibody, or antigen-binding fragment thereof, maycomprise a heavy chain variable region having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 360 and a light chain variable region having anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO: 361.

In some embodiments, the heavy chain variable region and/or the lightchain variable region sequences do not vary within any of the CDRsequences provided herein. For example, in some embodiments, the degreeof sequence variation (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) may occurwithin a heavy chain variable and/or a light chain variable amino acidsequence excluding any of the CDR sequences provided herein. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 7 and a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 8. In some embodiments, theantibody, or antigen-binding fragment thereof, comprising a heavy chainvariable region having an amino acid sequence that is at least 90%identical to SEQ ID NO: 318 and/or a light chain variable region havingan amino acid sequence that is at least 90% identical to SEQ ID NO: 319does not vary within any of the CDR sequences of Ab42 provided herein.In some embodiments, the antibody, or antigen-binding fragment thereof,comprising a heavy chain variable region having an amino acid sequencethat is at least 90% identical to SEQ ID NO: 360 and/or a light chainvariable region having an amino acid sequence that is at least 90%identical to SEQ ID NO: 361 does not vary within any of the CDRsequences of Ab63 provided herein.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in Table 6, and/or a light chain variable domaincomprising an amino acid sequence set forth in Table 6. For example, insome embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 6 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 7. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 6 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 7. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 6 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 7.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 74 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 75. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 74 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 75. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 74 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 75.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 76 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 77. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 76 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 77. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 76 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 77.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 78 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 79. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 78 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 79. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 78 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 79.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 80 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 81. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 80 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 81. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 80 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 81.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 82 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 83. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 82 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 83. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 82 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 83.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 84 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 85. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 84 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 85. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 84 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 85.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 86 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 87. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 86 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 87. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 86 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 87.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 88 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 89. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 88 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 89. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 88 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 89.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 90 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 91. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 90 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 91. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 90 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 91.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 92 and/or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 93. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 92 or a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 93. In some embodiments, theantibody, or antigen-binding portion thereof, comprises a heavy chainvariable domain comprising an amino acid sequence set forth in SEQ IDNO: 92 and a light chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 93.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 106 and/or a light chain variabledomain comprising an amino acid sequence set forth in SEQ ID NO: 107. Insome embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 106 or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 107. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 106 and a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 107.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 318 and/or a light chain variabledomain comprising an amino acid sequence set forth in SEQ ID NO: 319. Insome embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 318 or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 319. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 318 and a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 319.

In some embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 360 and/or a light chain variabledomain comprising an amino acid sequence set forth in SEQ ID NO: 361. Insome embodiments, the antibody, or antigen-binding portion thereof,comprises a heavy chain variable domain comprising an amino acidsequence set forth in SEQ ID NO: 360 or a light chain variable domaincomprising an amino acid sequence set forth in SEQ ID NO: 361. In someembodiments, the antibody, or antigen-binding portion thereof, comprisesa heavy chain variable domain comprising an amino acid sequence setforth in SEQ ID NO: 360 and a light chain variable domain comprising anamino acid sequence set forth in SEQ ID NO: 361.

The amino acid sequences of the heavy chain variable region (HCVR) andthe light chain variable region (LCVR) of the antibody SR-AB2 set forthin Table 5 are provided below.

SR-AB2 - Heavy chain variable region amino acid sequence (SEQ ID NO: 7)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARAP LGNFDSWGQGTMVTVSSSR-AB2 - Light chain variable region amino acid sequence (SEQ ID NO: 8)NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNHP VVFGGGTKLTVL

The amino acid sequences of the heavy chain variable region (HCVR) andthe light chain variable region (LCVR) of the antibody SR-AB10 set forthin Table 5 are provided below.

SR-AB10 - Heavy chain variable region amino acid sequence(SEQ ID NO: 88) QLQLQESGGGVVQPGRSLRLSCAASGFTFNNYPIHWVRQAPGKGLEWVAVMSYDGINKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPR IAARRGGFDYWGQGTLVTVSSSR-AB10 - Light chain variable region amino acid sequence(SEQ ID NO: 89) NFMLTQPHSVSESPGKTVTISCTRSSGNIDNNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSDNQG VVFGGGTKLTVL

The amino acid sequences of the heavy chain variable region (HCVR) andthe light chain variable region (LCVR) of the antibody SR-AB13 set forthin Table 5 are provided below.

SR-AB13 - Heavy chain variable region amino acid sequence(SEQ ID NO: 106) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSISYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDPSYDSIAGMDVWGQGTTVTVSS SR-AB13 - Light chain variable region amino acidsequence (SEQ ID NO: 107)DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFDFPFTFGG GTKVEIK

The amino acid sequences of the heavy chain variable region (HCVR) andthe light chain variable region (LCVR) of the antibody SR-AB42 set forthin Table 5 are provided below.

SR-AB42 - Heavy chain variable region amino acid sequence(SEQ ID NO: 318) QVQLVESGGGVVQPGRSLRLSCAASGFTFRSYVMHWVRQAPGKGLEWVAVISHEGSLKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPR IAARRGGFGYWGQGTLVTVSS63 - Light chain variable region amino acid sequence (SEQ ID NO: 319)NFMLTQPHSVSESPGKTVTISCTRSSGNIDNNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDYDTQG  VVFGGGTKLTVL

The amino acid sequences of the heavy chain variable region (HCVR) andthe light chain variable region (LCVR) of the antibody SR-AB63 set forthin Table 5 are provided below.

SR-AB63 - Heavy chain variable region amino acid sequence(SEQ ID NO: 360) QLQLQESGPGLVKPSETLSLTCTVSGGSIRSSSYYWGWIRQPPGKGLEWIGSISYSATTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAGDPSYDSIAGMQVWGQGTTVTVSS SR-AB63 - Light chain variable region amino acidsequence (SEQ ID NO: 361)DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFDWPLTFGG GTKVEIK

In some embodiments, antibodies, or antigen-binding portions thereof, ofthe invention that selectively bind to a LTBP1-TGFβ complex and/or aLTBP3-TGFβ complex have one or more CDR sequences substantially similarto CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3. For example, theantibodies may include one or more CDR sequences as shown in Table 5(SEQ ID NOs: 1-6, SEQ ID NOs: 94-99 or SEQ ID NOs: 100-105) containingup to 6, 5, 4, 3, 2, or 1 amino acid residue variations as compared tothe corresponding CDR region in any one of SEQ ID NOs: 1-6, SEQ ID NOs:94-99 or SEQ ID NOs: 100-105.

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises at least three CDRs selected from the following, optionallycomprising up to 6 amino acid changes, for example 1, 2, 3, 4, 5, or6amino acid changes, for each of the CDRs CDR-H1: SEQ ID NO: 1; CDR-H2:SEQ ID NO: 2; CDR-H3: SEQ ID NO: 3; CDR-L1: SEQ ID NO: 4; CDR-L2: SEQ IDNO: 5; and, CDR-L3: SEQ ID NO: 6. In one embodiment, the antibody, orantigen-binding fragment thereof, comprises at least three CDRs selectedfrom the following, optionally comprising up to 6 amino acid changes,for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRsCDR-H1: SEQ ID NO: 94; CDR-H2: SEQ ID NO: 95; CDR-H3: SEQ ID NO: 96;CDR-L1: SEQ ID NO: 97; CDR-L2: SEQ ID NO: 98; and, CDR-L3: SEQ ID NO:99. In one embodiment, the antibody, or antigen-binding fragmentthereof, comprises at least three CDRs selected from the following,optionally comprising up to 6 amino acid changes, for example 1, 2, 3,4, 5, or 6 amino acid changes, for each of the CDRs CDR-H1: SEQ ID NO:100; CDR-H2: SEQ ID NO: 101; CDR-H3: SEQ ID NO: 102; CDR-Ll: SEQ ID NO:103; CDR-L2: SEQ ID NO: 104; and, CDR-L3: SEQ ID NO: 105. In oneembodiment, the antibody, or antigen-binding fragment thereof, comprisesat least three CDRs selected from the following, optionally comprisingup to 6 amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acidchanges, for each of the CDRs CDR-H1: SEQ ID NO: 166; CDR-H2: SEQ ID NO:167; CDR-H3: SEQ ID NO: 168; CDR-Ll: SEQ ID NO: 169; CDR-L2: SEQ ID NO:170; and, CDR-L3: SEQ ID NO: 171. In one embodiment, the antibody, orantigen-binding fragment thereof, comprises at least three CDRs selectedfrom the following, optionally comprising up to 6 amino acid changes,for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRsCDR-H1: SEQ ID NO: 292; CDR-H2: SEQ ID NO: 293; CDR-H3: SEQ ID NO: 294;CDR-L1: SEQ ID NO: 295; CDR-L2: SEQ ID NO: 296; and, CDR-L3: SEQ ID NO:297.

In one aspect, the invention provides an antibody, or antigen-bindingfragment thereof, comprising a heavy chain variable region comprisingCDR-H1: SEQ ID NO: 1; CDR-H2: SEQ ID NO: 2; and CDR-H3: SEQ ID NO: 3;and a light chain variable region comprising CDR-L1: SEQ ID NO: 4;CDR-L2: SEQ ID NO: 5; and CDR-L3: SEQ ID NO: 6, optionally comprisingone or more amino acid changes, for example 1, 2, 3, 4, 5, or 6 aminoacid changes, for each of the CDRs.

In one aspect, the invention provides an antibody, or antigen-bindingfragment thereof, comprising a heavy chain variable region comprisingCDR-H1: SEQ ID NO: 94; CDR-H2: SEQ ID

NO: 95; and CDR-H3: SEQ ID NO: 96; and a light chain variable regioncomprising CDR-L1: SEQ ID NO: 97; CDR-L2: SEQ ID NO: 98; and CDR-L3: SEQID NO: 99, optionally comprising one or more amino acid changes, forexample 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRs.

In one aspect, the invention provides an antibody, or antigen-bindingfragment thereof, comprising a heavy chain variable region comprisingCDR-H1: SEQ ID NO: 100; CDR-H2: SEQ ID NO: 101; and CDR-H3: SEQ ID NO:102; and a light chain variable region comprising CDR-Ll: SEQ ID NO:103; CDR-L2: SEQ ID NO: 104; and CDR-L3: SEQ ID NO: 105, optionallycomprising one or more amino acid changes, for example 1, 2, 3, 4, 5, or6 amino acid changes, for each of the CDRs.

In one aspect, the invention provides an antibody, or antigen-bindingfragment thereof, comprising a heavy chain variable region comprisingCDR-H1: SEQ ID NO: 166; CDR-H2: SEQ ID NO: 167; and CDR-H3: SEQ ID NO:168; and a light chain variable region comprising CDR-L1: SEQ ID NO:169; CDR-L2: SEQ ID NO: 170; and CDR-L3: SEQ ID NO: 171, optionallycomprising one or more amino acid changes, for example 1, 2, 3, 4, 5, or6 amino acid changes, for each of the CDRs. For instance, if there arechanges within the CDRs, there may be up to 1 change per CDR. There maybe no more than 2 changes across all 6 CDRs.

In one aspect, the invention provides an antibody, or antigen-bindingfragment thereof, comprising a heavy chain variable region comprisingCDR-H1: SEQ ID NO: 292; CDR-H2: SEQ ID NO: 293; and CDR-H3: SEQ ID NO:294; and a light chain variable region comprising CDR-L1: SEQ ID NO:295; CDR-L2: SEQ ID NO: 296; and CDR-L3: SEQ ID NO: 297, optionallycomprising one or more amino acid changes, for example 1, 2, 3, 4, 5, or6 amino acid changes (e.g., up to 2), for each of the CDRs. Forinstance, if there are changes within the CDRs, there may be up to 1change per CDR. There may be no more than 2 changes across all 6 CDRs.

In one aspect, the invention provides an antibody, or antigen-bindingfragment thereof comprising a heavy chain variable region comprising aCDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 having particularamino acid changes. As used herein, the phrase “amino acid changes” or“changes in amino acid residues” includes amino acid substitutions,additions, and/or deletions. In some embodiments, there are one or morechanges to the amino acid residues with any one of the CDRs and/orvariable regions described herein. For example, in some embodiments, theone or more amino acid changes comprises one amino acid change. In someembodiments, the one or more amino acid changes comprises up to twoamino acid changes. In some embodiments, the one or more amino acidchanges comprises up to three amino acid changes. In some embodiments,the one or more amino acid changes comprises up to four amino acidchanges. In some embodiments, the one or more amino acid changescomprises up to five amino acid changes. In some embodiments, the one ormore amino acid changes comprises up to six amino acid changes. In someembodiments, the one or more amino acid changes comprises up to sevenamino acid changes.

For example, in some embodiments, the antibody, or antigen-bindingfragment thereof, comprises a CDR-H1: SEQ ID NO:1, with the proviso thatthe threonine residue at position 4 of SEQ ID NO:1 may be substitutedwith a histidine, lysine, phenylalanine, or glycine. In someembodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:1, with the proviso that the serineresidue at position 5 of SEQ ID NO:1 may be substituted with a leucine.In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:1, with the proviso that the serineresidue at position 9 of SEQ ID NO:1 may be substituted with an alanine.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:1, with the proviso that (i) the threonineresidue at position 4 of SEQ ID NO:1 may be substituted with ahistidine, lysine, phenylalanine, or glycine; (ii) the serine residue atposition 5 of SEQ ID NO:1 may be substituted with a leucine; and/or,(iii) the serine residue at position 9 of SEQ ID NO:1 may be substitutedwith an alanine.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H2: SEQ ID NO:2, with the proviso that the serineresidue at position 3 of SEQ ID NO:2 may be substituted with anaspartate or asparagine. In some embodiments, the antibody, orantigen-binding fragment thereof, comprises a CDR-H2: SEQ ID NO:2, withthe proviso that the tyrosine residue at position 5 of SEQ ID NO:2 maybe substituted with a histidine. In some embodiments, the antibody, orantigen-binding fragment thereof, comprises a CDR-H2: SEQ ID NO:2, withthe proviso that the asparagine residue at position 6 of SEQ ID NO:2 maybe substituted with a serine. In some embodiments, the antibody, orantigen-binding fragment thereof, comprises a CDR-H2: SEQ ID NO:2, withthe proviso that the asparagine residue at position 8 of SEQ ID NO:2 maybe substituted with a phenylalanine, leucine, alanine, tyrosine,aspartate, or serine. In some embodiments, the antibody, orantigen-binding fragment thereof, comprises a CDR-H2: SEQ ID NO:2, withthe proviso that the asparagine residue at position 10 of SEQ ID NO:2may be substituted with an aspartate or alanine.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H2: SEQ ID NO:2, with the proviso that (i) the serineresidue at position 3 of SEQ ID NO:2 may be substituted with anaspartate or asparagine; (ii) the tyrosine residue at position 5 of SEQID NO:2 may be substituted with a histidine; (iii) the asparagineresidue at position 6 of SEQ ID NO:2 may be substituted with a serine;(iv) the asparagine residue at position 8 of SEQ ID NO:2 may besubstituted with a phenylalanine, leucine, alanine, tyrosine, aspartate,or serine; and/or, (v) the asparagine residue at position 10 of SEQ IDNO:2 may be substituted with a aspartate or alanine.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises three heavy chain CDRs and three light chain CDRs, wherein theheavy chain CDRs comprise:

a) CDR-H1: SEQ ID NO:1, with the proviso that:

-   -   i. the threonine residue at position 4 of SEQ ID NO:1 may be        substituted with a histidine, lysine, phenylalanine, or glycine;    -   ii. the serine residue at position 5 of SEQ ID NO:1 may be        substituted with a leucine; and/or,    -   iii. the serine residue at position 9 of SEQ ID NO:1 may be        substituted with an alanine;

b) CDR-H2: SEQ ID NO:2, with the proviso that:

-   -   i. the serine residue at position 3 of SEQ ID NO:2 may be        substituted with an aspartate or asparagine;    -   ii. the tyrosine residue at position 5 of SEQ ID NO:2 may be        substituted with a histidine;    -   iii. the asparagine residue at position 6 of SEQ ID NO:2 may be        substituted with a serine;    -   iv. the asparagine residue at position 8 of SEQ ID NO:2 may be        substituted with a phenylalanine, leucine, alanine, tyrosine,        aspartate, or serine; and/or,    -   v. the asparagine residue at position 10 of SEQ ID NO:2 may be        substituted with a aspartate or alanine;

c) CDR-H3: SEQ ID NO:3, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment thereof,further comprises a CDR-L1 as set forth in SEQ ID NO:4, optionallycomprising one or more amino acid changes. In some embodiments, theantibody, or antigen-binding fragment thereof, further comprises aCDR-L2 as set forth in SEQ ID NO:5, optionally comprising one or moreamino acid changes. In some embodiments, the antibody, orantigen-binding fragment thereof, further comprises a CDR-L3 as setforth in SEQ ID NO:6, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises at least three of the following six CDRs:

CDR-H1: SEQ ID NO:1, with the proviso that:

-   -   i. the threonine residue at position 4 of SEQ ID NO:1 may be        substituted with a histidine, lysine, phenylalanine, or glycine;    -   ii. the serine residue at position 5 of SEQ ID NO:1 may be        substituted with an leucine; and/or,    -   iii. the serine residue at position 9 of SEQ ID NO:1 may be        substituted with an alanine;

b) CDR-H2: SEQ ID NO:2, with the proviso that:

-   -   i. the serine residue at position 3 of SEQ ID NO:2 may be        substituted with an aspartate or asparagine;    -   ii. the tyrosine residue at position 5 of SEQ ID NO:2 may be        substituted with a histidine;    -   iii. the asparagine residue at position 6 of SEQ ID NO:2 may be        substituted with a serine;    -   iv. the asparagine residue at position 8 of SEQ ID NO:2 may be        substituted with a phenylalanine, leucine, alanine, tyrosine,        aspartate, or serine; and/or,    -   v. the asparagine residue at position 10 of SEQ ID NO:2 may be        substituted with a aspartate or alanine;

c) CDR-H3: SEQ ID NO:3, optionally comprising one or more amino acidchanges;

d) CDR-L1: SEQ ID NO:4, optionally comprising one or more amino acidchanges;

e) CDR-L2: SEQ ID NO:5, optionally comprising one or more amino acidchanges; and,

f) CDR-L3: SEQ ID NO:6, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment thereof,specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex.In some embodiments, the antibody, or antigen-binding fragment thereof,does not bind a human GARP-proTGFβ1 complex. In some embodiments, theantibody, or antigen-binding fragment thereof, does not bind matureTGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the invention provides an isolated antibodythat specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complexor a human LRRC33-proTGFβ1 complex; wherein the antibody does not bindmature TGFβ1, mature TGFβ2 or mature TGFβ33; wherein the antibody is afully human or humanized antibody or a fragment thereof, and wherein theantibody comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:1, with the proviso that:

-   -   i. the threonine residue at position 4 of SEQ ID NO:1 may be        substituted with a histidine, lysine, phenylalanine, or glycine;    -   ii. the serine residue at position 5 of SEQ ID NO:1 may be        substituted with an leucine; and/or,    -   iii. the serine residue at position 9 of SEQ ID NO:1 may be        substituted with an alanine;

b) CDR-H2: SEQ ID NO:2, with the proviso that:

-   -   i. the serine residue at position 3 of SEQ ID NO:2 may be        substituted with an aspartate or asparagine;    -   ii. the tyrosine residue at position 5 of SEQ ID NO:2 may be        substituted with a histidine;    -   iii. the asparagine residue at position 6 of SEQ ID NO:2 may be        substituted with a serine;    -   iv. the asparagine residue at position 8 of SEQ ID NO:2 may be        substituted with a phenylalanine, leucine, alanine, tyrosine,        aspartate, or serine; and/or,    -   v. the asparagine residue at position 10 of SEQ ID NO:2 may be        substituted with a aspartate or alanine;

c) CDR-H3: SEQ ID NO:3, optionally comprising one or more amino acidchanges;

d) CDR-L1: SEQ ID NO:4, optionally comprising one or more amino acidchanges;

e) CDR-L2: SEQ ID NO:5, optionally comprising one or more amino acidchanges; and,

f) CDR-L3: SEQ ID NO:6, optionally comprising one or more amino acidchanges.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <100 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <100 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <50 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <50 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <25 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <25 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <10 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <10 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouseLTBP1-proTGFβ1. In some embodiments, such antibody is alsocross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a K_(D) of <100 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a K_(D) of <100 nM as measured in a suitable in vitrobinding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <50 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <50 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <25 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <25 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <10 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <10 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to humanGARP-proTGFβ1. In preferred embodiments, such context-selective antibodyis isoform-specific in that it selectively binds and inhibits theactivation of TGFβ1 associated with LTBP1/3 and does not bind to humanGARP-proTGFβ1.

In another aspect, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:94, with the proviso that the threonineresidue at position 2 of SEQ ID NO:94 may be substituted with analanine. In some embodiments, the antibody, or antigen-binding fragmentthereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that theasparagine residue at position 4 of SEQ ID NO:94 may be substituted withan alanine, tyrosine, aspartate, serine, arginine, or histidine. In someembodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:94, with the proviso that the asparagineresidue at position 5 of SEQ ID NO:94 may be substituted with aglutamine, serine, glycine, lysine, glutamate, arginine, or histidine.In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:94, with the proviso that the tyrosineresidue at position 6 of SEQ ID NO:94 may be substituted with aarginine. In some embodiments, the antibody, or antigen-binding fragmentthereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that theproline residue at position 7 of SEQ ID NO:94 may be substituted with aglycine, alanine, leucine, serine, asparagine, valine, aspartate, orglutamine In some embodiments, the antibody, or antigen-binding fragmentthereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that theisoleucine residue at position 8 of SEQ ID NO:94 may be substituted witha methionine or leucine. In some embodiments, the antibody, orantigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:94, withthe proviso that the histidine residue at position 9 of SEQ ID NO:94 maybe substituted with a phenylalanine, tyrosine, asparagine, or serine.SEQ ID NO: 94 comprising these substitutions is disclosed as SEQ ID NO:399.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:94, with the proviso that (i) thethreonine residue at position 2 of SEQ ID NO:94 may be substituted withan alanine; (ii) the asparagine residue at position 4 of SEQ ID NO:94may be substituted with an alanine, tyrosine, aspartate, serine,arginine, or histidine; (iii) the asparagine residue at position 5 ofSEQ ID NO:94 may be substituted with a glutamine, serine, glycine,lysine, glutamate, arginine, or histidine; (iv) the tyrosine residue atposition 6 of SEQ ID NO:94 may be substituted with a arginine; (v) theproline residue at position 7 of SEQ ID NO:94 may be substituted with aglycine, alanine, leucine, serine, asparagine, valine, aspartate, orglutamine; (vi) the isoleucine residue at position 8 of SEQ ID NO:94 maybe substituted with a methionine or leucine; and/or, (vii) the histidineresidue at position 9 of SEQ ID NO:94 may be substituted with aphenylalanine, tyrosine, asparagine, or serine. SEQ ID NO: 94 comprisingthese substitutions is disclosed as SEQ ID NO: 399.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises three heavy chain CDRs and three light chain CDRs, wherein theheavy chain CDRs comprise:

a) CDR-H1: SEQ ID NO:94 (SEQ ID NO: 94 comprising these substitutions isdisclosed as SEQ ID NO: 399), with the proviso that:

-   -   i. the threonine residue at position 2 of SEQ ID NO:94 may be        substituted with an alanine;    -   ii. the asparagine residue at position 4 of SEQ ID NO:94 may be        substituted with an alanine, tyrosine, aspartate, serine,        arginine, or histidine;    -   iii. the asparagine residue at position 5 of SEQ ID NO:94 may be        substituted with a glutamine, serine, glycine, lysine,        glutamate, arginine, or histidine;    -   iv. the tyrosine residue at position 6 of SEQ ID NO:94 may be        substituted with a arginine;    -   v. the proline residue at position 7 of SEQ ID NO:94 may be        substituted with a glycine, alanine, leucine, serine,        asparagine, valine, aspartate, or glutamine;    -   vi. the isoleucine residue at position 8 of SEQ ID NO:94 may be        substituted with a methionine or leucine; and/or,    -   vii. the histidine residue at position 9 of SEQ ID NO:94 may be        substituted with a phenylalanine, tyrosine, asparagine, or        serine;

b) CDR-H2: SEQ ID NO:95, optionally comprising one or more amino acidchanges; and

c) CDR-H3: SEQ ID NO:96, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment thereof,further comprises a CDR-L1 as set forth in SEQ ID NO:97, optionallycomprising one or more amino acid changes. In some embodiments, theantibody, or antigen-binding fragment thereof, further comprises aCDR-L2 as set forth in SEQ ID NO:98, optionally comprising one or moreamino acid changes. In some embodiments, the antibody, orantigen-binding fragment thereof, further comprises a CDR-L3 as setforth in SEQ ID NO:99, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:94 (SEQ ID NO: 94 comprising these substitutions isdisclosed as SEQ ID NO: 399), with the proviso that:

-   -   i. the threonine residue at position 2 of SEQ ID NO:94 may be        substituted with an alanine;    -   ii. the asparagine residue at position 4 of SEQ ID NO:94 may be        substituted with an alanine, tyrosine, aspartate, serine,        arginine, or histidine;    -   iii. the asparagine residue at position 5 of SEQ ID NO:94 may be        substituted with a glutamine, serine, glycine, lysine,        glutamate, arginine, or histidine;    -   iv. the tyrosine residue at position 6 of SEQ ID NO:94 may be        substituted with a arginine;    -   v. the proline residue at position 7 of SEQ ID NO:94 may be        substituted with a glycine, alanine, leucine, serine,        asparagine, valine, aspartate, or glutamine;    -   vi. the isoleucine residue at position 8 of SEQ ID NO:94 may be        substituted with a methionine or leucine; and/or,    -   vii. the histidine residue at position 9 of SEQ ID NO:94 may be        substituted with a phenylalanine, tyrosine, asparagine, or        serine;

b) CDR-H2: SEQ ID NO:95, optionally comprising one or more amino acidchanges;

c) CDR-H3: SEQ ID NO:96, optionally comprising one or more amino acidchanges;

d) CDR-L1: SEQ ID NO:97, optionally comprising one or more amino acidchanges;

e) CDR-L2: SEQ ID NO:98, optionally comprising one or more amino acidchanges; and,

f) CDR-L3: SEQ ID NO:99, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment thereof,specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex.In some embodiments, the antibody, or antigen-binding fragment thereof,does not bind a human GARP-proTGFβ1 complex. In some embodiments, theantibody, or antigen-binding fragment thereof, does not bind matureTGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragmentthereof, specifically binds a human LTBP1-proTGFβ1 complex and/or ahuman LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1complex; wherein the antibody does not bind mature TGFβ1, mature TGFβ2or mature TGFβ33; wherein the antibody is a fully human or humanizedantibody or a fragment thereof, and wherein the antibody comprises atleast three of the following six CDRs:

a) CDR-H1: SEQ ID NO:94 (SEQ ID NO: 94 comprising these substitutions isdisclosed as SEQ ID NO: 399), with the proviso that:

-   -   i. the threonine residue at position 2 of SEQ ID NO:94 may be        substituted with an alanine;    -   ii. the asparagine residue at position 4 of SEQ ID NO:94 may be        substituted with an alanine, tyrosine, aspartate, serine,        arginine, or histidine;    -   iii. the asparagine residue at position 5 of SEQ ID NO:94 may be        substituted with a glutamine, serine, glycine, lysine,        glutamate, arginine, or histidine;    -   iv. the tyrosine residue at position 6 of SEQ ID NO:94 may be        substituted with a arginine;    -   v. the proline residue at position 7 of SEQ ID NO:94 may be        substituted with a glycine, alanine, leucine, serine,        asparagine, valine, aspartate, or glutamine;    -   vi. the isoleucine residue at position 8 of SEQ ID NO:94 may be        substituted with a methionine or leucine; and/or,    -   vii. the histidine residue at position 9 of SEQ ID NO:94 may be        substituted with a phenylalanine, tyrosine, asparagine, or        serine;

b) CDR-H2: SEQ ID NO:95, optionally comprising one or more amino acidchanges;

c) CDR-H3: SEQ ID NO:96, optionally comprising one or more amino acidchanges;

d) CDR-L1: SEQ ID NO:97, optionally comprising one or more amino acidchanges;

e) CDR-L2: SEQ ID NO:98, optionally comprising one or more amino acidchanges; and,

f) CDR-L3: SEQ ID NO:99, optionally comprising one or more amino acidchanges.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <100 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <100 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <50 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <50 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <25 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <25 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <10 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <10 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouseLTBP1-proTGFβ1. In some embodiments, such antibody is alsocross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a K_(D) of <100 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a K_(D) of <100 nM as measured in a suitable in vitrobinding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <50 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <50 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <25 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <25 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <10 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <10 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to humanGARP-proTGFβ1. In preferred embodiments, such context-selective antibodyis also isoform-specific in that it selectively binds and inhibits theactivation of TGFβ1 associated with LTBP1/3.

In another aspect, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1 comprising the amino acid sequence FTF(X₁)(X₂)YVMH,wherein, optionally: X₁ is S or R; and X₂ is G or S (SEQ ID NO: 392). Insome embodiments, X₁ is S. In some embodiments, X₁ is R. In someembodiments, X₂ is G. In some embodiments, X₂ is S.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H2 comprising the amino acid sequence(X₁)ISHEG(X₂)(X₃)KYYADSVKG, wherein, optionally: X₁ is V or S; X₂ is Sor G; and X₃ is F or L (SEQ ID NO: 393). In some embodiments, X₁ is a V.In some embodiment, X₁ is a S. In some embodiments, X₂ is S. In someembodiments, X₂ is G. In some embodiments, X₃ is F. In some embodiments,X₃ is L.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H3 comprising the amino acid sequence(X₁)(X₂)P(X₃)(X₄)(X₅)(X₆)RRGG(X₇)(X₈)(X₉), wherein, optionally: X₁ is Aor V; X₂ is R, V, G or K; X₃ is R, H or L; X₄ is I, V or G; X₅ is A, S,or L; X₆ is A or V; X₇ is F or Y; Xs is D, G, R, or S; and, X₉ is Y, G,R, L, V, A or K (SEQ ID NO: 394). In some embodiments, X₁ is A. In someembodiments, X₁ is V. In some embodiments, X₂ is R. In some embodiments,X₂ is V. In some embodiments, X₂ is G. In some embodiments, X₂ is K. Insome embodiments, X₃ is R. In some embodiments, X₃ is H. In someembodiments, X₃ is L. In some embodiments, X₄ is I. In some embodiments,X₄ is V. In some embodiments, X₄ is G. In some embodiments, X₅ is A. Insome embodiments, X₅ is S. In some embodiments, X₅ is L. In someembodiments, X₆ is A. In some embodiments, X₆ is V. In some embodiments,X₇ is F. In some embodiments, X₇ is Y. In some embodiments, X₈ is D. Insome embodiments, X₈ is G. In some embodiments, X₈ is R. In someembodiments, X₈ is S. In some embodiments, X₉ is Y. In some embodiments,X₉ is G. In some embodiments, X₉ is R. In some embodiments, X₉ is L. Insome embodiments, X₉ is V. In some embodiments, X₉ is A. In someembodiments, X₉ is K.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises three heavy chain CDRs and three light chain CDRs, wherein theheavy chain CDRs comprise:

a) CDR-H1 comprising the amino acid sequence FTF(X₁)(X₂)YVMH, wherein,optionally: X₁ is S or R; and X₂ is G or S (SEQ ID NO: 392);

b) CDR-H2 comprising the amino acid sequence (X₁)ISHEG(X₂)(X₃)KYYADSVKG,wherein, optionally: X₁ is V or S; X₂ is S or G; and X₃ is F or L (SEQID NO: 393); and c) CDR-H3 comprising the amino acid sequence(X₁)(X₂)P(X₃)(X₄)(X₅)(X₆)RRGG(X₇) (X₈)(X₉), wherein, optionally: X₁ is Aor V; X₂ is R, V, G or K; X₃ is R, H or L; X₄ is I, V or G; X₅ is A, S,or L; X₆ is A or V; X₇ is F or Y; X₈ is D, G, R, or S; and, X₉ is Y, G,R, L, V, A or K (SEQ ID NO: 394).

In some embodiments, the antibody, or antigen-binding fragment thereof,further comprises a CDR-L1 as set forth in SEQ ID NO:97, optionallycomprising one or more amino acid changes. In some embodiments, theantibody, or antigen-binding fragment thereof, further comprises aCDR-L2 as set forth in SEQ ID NO:98, optionally comprising one or moreamino acid changes. In some embodiments, the antibody, orantigen-binding fragment thereof, further comprises a CDR-L3 as setforth in SEQ ID NO:99, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment,comprises at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTF(X₁)(X₂)YVMH, wherein,optionally: X₁ is S or R; and X₂ is G or S (SEQ ID NO: 392);

b) CDR-H2 comprising the amino acid sequence (X₁)ISHEG(X₂)(X₃)KYYADSVKG,wherein, optionally: X₁ is V or S; X₂ is S or G; and X₃ is F or L (SEQID NO: 393);

c) CDR-H3 comprising the amino acid sequence(X₁)(X₂)P(X₃)(X₄)(X₅)(X₆)RRGG(X₇) (X₈)(X₉), wherein, optionally: X₁ is Aor V; X₂ is R, V, G or K; X₃ is R, H or L; X₄ is I, V or G; X₅ is A, S,or L; X₆ is A or V; X₇ is F or Y; X₈ is D, G, R, or S; and, X₉ is Y, G,R, L, V, A or K (SEQ ID NO: 394);

d) CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one ormore amino acid changes;

e) CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one ormore amino acid changes; and

f) CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one ormore amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof,specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex.In some embodiments, the antibody, or antigen-binding fragment thereof,does not bind a human GARP-proTGFβ1 complex. In some embodiments, theantibody, or antigen-binding fragment thereof, does not bind matureTGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragment,specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex;wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or matureTGFβ33; wherein the antibody is a fully human or humanized antibody or afragment thereof, wherein the antibody comprises at least three of thefollowing six CDRs:

a) CDR-H1 comprising the amino acid sequence FTF(X₁)(X₂)YVMH, wherein,optionally: X₁ is S or R; and X₂ is G or S (SEQ ID NO: 392);

b) CDR-H2 comprising the amino acid sequence (X₁)ISHEG(X₂)(X₃)KYYADSVKG,wherein, optionally: X₁ is V or S; X₂ is S or G; and X₃ is F or L (SEQID NO: 393);

c) CDR-H3 comprising the amino acid sequence(X₁)(X₂)P(X₃)(X₄)(X₅)(X₆)RRGG(X₇) (X₈)(X₉), wherein, optionally: X₁ is Aor V; X₂ is R, V, G or K; X₃ is R, H or L; X₄ is I, V or G; X₅ is A, S,or L; X₆ is A or V; X₇ is F or Y; X₈ is D, G, R, or S; and, X₉ is Y, G,R, L, V, A or K (SEQ ID NO: 394);

d) CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one ormore amino acid changes;

e) CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one ormore amino acid changes; and

f) CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one ormore amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <100 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <100 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <50 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <50 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <25 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <25 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <10 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <10 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouseLTBP1-proTGFβ1. In some embodiments, such antibody is alsocross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a K_(D) of <100 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a K_(D) of <100 nM as measured in a suitable in vitrobinding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <50 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <50 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <25 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <25 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <10 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <10 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to humanGARP-proTGFβ1. In preferred embodiments, such context-selective antibodyis also isoform-specific in that it selectively binds and inhibits theactivation of TGFβ1 associated with LTBP1/3.

In another aspect, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1 comprising the amino acid sequence FTF(X₁)(X₂)YVMH,wherein, optionally: X₁ is S or R; and X₂ is G or S (SEQ ID NO: 392). Insome embodiments, X₁ is S. In some embodiments, X₁ is R. In someembodiments, X₂ is G. In some embodiments, X₂ is S.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H2 comprising the amino acid sequence(X₁)ISHEGS(X₂)KYYADSVKG, wherein, optionally: X₁ is V or S; and, X₂ is For L (SEQ ID NO: 382). In some embodiments, X₁ is a V. In someembodiment, X₁ is a S. In some embodiments, X₃ is F. In someembodiments, X₃ is L.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H3 comprising the amino acid sequenceA(X₁)PRI(X₂)ARRGGFGY, wherein, optionally: X₁ is R or V; X₂ is A or L(SEQ ID NO: 383). In some embodiments, X₁ is R. In some embodiments, X₁is V. In some embodiments, X₂ is A. In some embodiments, X₂ is L.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises three heavy chain CDRs and three light chain CDRs, wherein theheavy chain CDRs comprise:

a) CDR-H1 comprising the amino acid sequence FTF(X₁)(X₂)YVMH, wherein,optionally: X₁ is S or R; and X₂ is G or S (SEQ ID NO: 392);

b) CDR-H2 comprising the amino acid sequence (X₁)ISHEGS(X₂)KYYADSVKG,wherein, optionally: X₁ is V or S; and, X₂ is F or L (SEQ ID NO: 382);and

c) CDR-H3 comprising the amino acid sequence A(X₁)PRI(X₂)ARRGGFGY,wherein, optionally: X₁ is R or V; X₂ is A or L (SEQ ID NO: 383).

In some embodiments, the antibody, or antigen-binding fragment thereof,further comprises a CDR-L1 as set forth in SEQ ID NO:97, optionallycomprising one or more amino acid changes. In some embodiment, theantibody, or antigen-binding fragment thereof, further comprises aCDR-L2 as set forth in SEQ ID NO:98, optionally comprising one or moreamino acid changes. In some embodiments, the antibody, orantigen-binding fragment thereof, further comprises a CDR-L3 as setforth in SEQ ID NO:99, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment,comprises at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTF(X₁)(X₂)YVMH, wherein,optionally: X₁ is S or R; and X₂ is G or S (SEQ ID NO: 392);

b) CDR-H2 comprising the amino acid sequence (X₁)ISHEGS(X₂)KYYADSVKG,wherein, optionally: X₁ is V or S; and, X₂ is F or L (SEQ ID NO: 382);

c) CDR- comprising the amino acid sequence A(X₁)PRI(X₂)ARRGGFGY,wherein, optionally: X₁ is R or V; X₂ is A or L (SEQ ID NO: 383);

d) CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one ormore amino acid changes;

e) CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one ormore amino acid changes; and

f) CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one ormore amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof,specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex.In some embodiments, the antibody, or antigen-binding fragment thereof,does not bind a human GARP-proTGFβ1 complex. In some embodiments, theantibody, or antigen-binding fragment thereof, does not bind matureTGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragmentthereof, specifically binds a human LTBP1-proTGFβ1 complex and/or ahuman LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1complex; wherein the antibody does not bind mature TGFβ1, mature TGFβ2or mature TGFβ33; wherein the antibody is a fully human or humanizedantibody or a fragment thereof, wherein the antibody comprises at leastthree of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTF(X₁)(X₂)YVMH, wherein,optionally: X₁ is S or R; and X₂ is G or S (SEQ ID NO: 392);

b) CDR-H2 comprising the amino acid sequence (X₁)ISHEGS(X₂)KYYADSVKG,wherein: X₁ is V or S; and, X₂ is F or L (SEQ ID NO: 382);

c) CDR-H3 comprising the amino acid sequence A(X₁)PRI(X₂)ARRGGFGY,wherein, optionally: X₁ is R or V; X₂ is A or L (SEQ ID NO: 383);

d) CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one ormore amino acid changes;

e) CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one ormore amino acid changes; and

f) CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one ormore amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <100 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <100 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <50 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <50 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a K_(D) of <25 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a K_(D) of <25 nM as measured in asuitable in vitro binding assay such as Bio-Layer

Interferometry (BLI). In some embodiments, such antibody binds a humanLTBP1-proTGFβ1 complex with a K_(D) of <10 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI); and/or,such antibody binds a human LTBP3-proTGFβ1 complex with a K_(D) of <10nM as measured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouseLTBP1-proTGFβ1. In some embodiments, such antibody is alsocross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a K_(D) of <100 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a K_(D) of <100 nM as measured in a suitable in vitrobinding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <50 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <50 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <25 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <25 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI). In someembodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with aK_(D) of <10 nM as measured in a suitable in vitro binding assay such asBio-Layer Interferometry (BLI); and/or, the antibody binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <10 nM as measured in a suitablein vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to humanGARP-proTGFβ1. In preferred embodiments, such context-selective antibodyis also isoform-specific in that it selectively binds and inhibits theactivation of TGFβ1 associated with LTBP1/3.

In another aspect, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:100, with the proviso that the serineresidue at position 4 of SEQ ID NO:100 may be substituted with ahistidine. In some embodiments, the antibody, or antigen-bindingfragment thereof, comprises a CDR-H1: SEQ ID NO:100, with the provisothat the serine residue at position 7 of SEQ ID NO:100 may besubstituted with an alanine or glycine. In some embodiments, theantibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQID NO:100, with the proviso that the glycine residue at position 11 ofSEQ ID NO:100 may be substituted with a threonine, serine, histidine,leucine, isoleucine, asparagine, valine, or alanine. SEQ ID NO: 100comprising these substitutions is disclosed as SEQ ID NO: 400.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:100, with the proviso that (i) the serineresidue at position 4 of SEQ ID NO:100 may be substituted with ahistidine; (ii) the serine residue at position 7 of SEQ ID NO:100 may besubstituted with an alanine or glycine; and/or, (iii) the glycineresidue at position 11 of SEQ ID NO:100 may be substituted with athreonine, serine, histidine, leucine, isoleucine, asparagine, valine,or alanine. SEQ ID NO: 100 comprising these substitutions is disclosedas SEQ ID NO: 400.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:101, with the proviso that the serineresidue at position 3 of SEQ ID NO:101 may be substituted with analanine. In some embodiments, the antibody, or antigen-binding fragmentthereof, comprises a CDR-H1: SEQ ID NO:101, with the proviso that theglycine residue at position 6 of SEQ ID NO:101 may be substituted withan alanine or serine. In some embodiments, the antibody, orantigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:101,with the proviso that the serine residue at position 7 of SEQ ID NO:101may be substituted with a threonine. SEQ ID NO: 101 comprising thesesubstitutions is disclosed as SEQ ID NO: 401.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1: SEQ ID NO:101, with the proviso that (i) the serineresidue at position 3 of SEQ ID NO:101 may be substituted with analanine; (ii) the glycine residue at position 6 of SEQ ID NO:101 may besubstituted with an alanine or serine; and/or, (iii) the serine residueat position 7 of SEQ ID NO:101 may be substituted with a threonine. SEQID NO: 101 comprising these substitutions is disclosed as SEQ ID NO:401.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises three heavy chain CDRs and three light chain CDRs, wherein theheavy chain CDRs comprise:

a) CDR-H1: SEQ ID NO:100 (SEQ ID NO: 100 comprising these substitutionsis disclosed as SEQ ID NO: 400), with the proviso that:

-   -   i. the serine residue at position 4 of SEQ ID NO:100 may be        substituted with a histidine;    -   ii. the serine residue at position 7 of SEQ ID NO:100 may be        substituted with an alanine or glycine; and/or,    -   iii. the glycine residue at position 11 of SEQ ID NO:100 may be        substituted with a threonine, serine, histidine, leucine,        isoleucine, asparagine, valine, or alanine;

h) CDR-H2: SEQ ID NO:101 (SEQ ID NO: 101 comprising these substitutionsis disclosed as SEQ ID NO: 401), with the proviso that:

-   -   i. the serine residue at position 3 of SEQ ID NO:101 may be        substituted with an alanine;    -   ii. the glycine residue at position 6 of SEQ ID NO:101 may be        substituted with an alanine or serine; and/or,    -   iii. the serine residue at position 7 of SEQ ID NO:101 may be        substituted with a threonine; and

c) CDR-H3: SEQ ID NO:102, optionally comprising up to three, four, fiveor six amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof,further comprises a CDR-L1 as set forth in SEQ ID NO:103, optionallycomprising one or more amino acid changes. In some embodiments, theantibody, or antigen-binding fragment thereof, further comprises aCDR-L2 as set forth in SEQ ID NO:104, optionally comprising one or moreamino acid changes. In some embodiments, the antibody, orantigen-binding fragment thereof, further comprises a CDR-L3 as setforth in SEQ ID NO:105, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:100 (SEQ ID NO: 100 comprising these substitutionsis disclosed as SEQ ID NO: 400), with the proviso that:

-   -   i. the serine residue at position 4 of SEQ ID NO:100 may be        substituted with a histidine;    -   ii. the serine residue at position 7 of SEQ ID NO:100 may be        substituted with an alanine or glycine; and/or,    -   iii. the glycine residue at position 11 of SEQ ID NO:100 may be        substituted with a threonine, serine, histidine, leucine,        isoleucine, asparagine, valine, or alanine;

b) CDR-H2: SEQ ID NO:101 (SEQ ID NO: 101 comprising these substitutionsis disclosed as SEQ ID NO: 401), with the proviso that:

-   -   i. the serine residue at position 3 of SEQ ID NO:101 may be        substituted with an alanine;    -   ii. the glycine residue at position 6 of SEQ ID NO:101 may be        substituted with an alanine or serine; and/or,    -   iii. the serine residue at position 7 of SEQ ID NO:101 may be        substituted with a threonine; and

c) CDR-H3: SEQ ID NO:102, optionally comprising one or more amino acidchanges.

d) CDR-L1: SEQ ID NO:103, optionally comprising one or more amino acidchanges;

e) CDR-L2: SEQ ID NO:104, optionally comprising one or more amino acidchanges; and,

f) CDR-L3: SEQ ID NO:105, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment,specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex.In some embodiments, the antibody, or antigen-binding fragment thereof,does not bind a human GARP-proTGFβ1 complex. In some embodiments, theantibody, or antigen-binding fragment thereof, does not bind matureTGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragment,specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex;wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or matureTGFβ33; wherein the antibody is a fully human or humanized antibody or afragment thereof, wherein the antibody comprises at least three of thefollowing six CDRs:

a) CDR-H1: SEQ ID NO:100 (SEQ ID NO: 100 comprising these substitutionsis disclosed as SEQ ID NO: 400), with the proviso that:

-   -   i. the serine residue at position 4 of SEQ ID NO:100 may be        substituted with a histidine;    -   ii. the serine residue at position 7 of SEQ ID NO:100 may be        substituted with an alanine or glycine; and/or,    -   iii. the glycine residue at position 11 of SEQ ID NO:100 may be        substituted with a threonine, serine, histidine, leucine,        isoleucine, asparagine, valine, or alanine;

b) CDR-H2: SEQ ID NO:101 (SEQ ID NO: 101 comprising these substitutionsis disclosed as SEQ ID NO: 401), with the proviso that:

-   -   i. the serine residue at position 3 of SEQ ID NO:101 may be        substituted with an alanine;    -   ii. the glycine residue at position 6 of SEQ ID NO:101 may be        substituted with an alanine or serine; and/or,    -   iii. the serine residue at position 7 of SEQ ID NO:101 may be        substituted with a threonine;

c) CDR-H3: SEQ ID NO:102, optionally comprising one or more amino acidchanges;

d) CDR-Ll: SEQ ID NO:103, optionally comprising one or more amino acidchanges;

e) CDR-L2: SEQ ID NO:104, optionally comprising one or more amino acidchanges; and,

f) CDR-L3: SEQ ID NO:105, optionally comprising one or more amino acidchanges.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <100 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <100 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <50 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <50 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <25 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <25 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <10 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <10 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouseLTBP1-proTGFβ1. In some embodiments, such antibody is alsocross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <100 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <100 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI). In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <50 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <50 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI). In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <25 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <25 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI). In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <10 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <10 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to humanGARP-proTGFβ1. In preferred embodiments, such context-selective antibodyis also isoform-specific in that it selectively binds and inhibits theactivation of TGFβ1 associated with LTBP1/3.

In another aspect, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1 comprising the amino acid sequenceG(X₁)I(X₂)S(X₃)SYYW(X₄), wherein, optionally: X₁ is S or P; X₂ is S, Hor R; X₃ is S or G; and, X₄ is G, I, N or V (SEQ ID NO: 395). In someembodiments, X₁ is a S. In some embodiments, X₁ is P. In someembodiments, X₂ is S. In some embodiments, X₂ is H. In some embodiments,X₂ is R. In some embodiments, X₃ is S. In some embodiments, X₃ is G. Insome embodiments, X₄ is G. In some embodiments, X₄ is I. In someembodiments, X₄ is N. In some embodiments, X₄ is V.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H2 comprising the amino acid sequenceSISYSA(X₁)TYYNPSLKS, wherein, optionally, X₁ is S or T (SEQ ID NO: 396).In some embodiments, X₁ is a S. In some embodiment, X₁ is a T.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H3 comprising the amino acid sequence(X₁)(X₂)D(X₃)(X₄)Y(X₅)(X₆)(X₇)(X₈)G(X₉)(X₁₀)(X₁₁), wherein, optionally:X₁ is A or V; X₂ is R, S or G, X₃ is P, Y, R, V, I, H, T or E; X₄ is S,D, E or N; X₅ is D, A or T; X₆ is S, G, T or A; X₇ is I, A, R, Q, or V;X₈ is A, E, K, G or T; X₉ is M or I; X₁₀ is D, L, Q, V, N or G; and, X₁i is V, R, N, E or K (SEQ ID NO: 397). In some embodiments, X₁ is A. Insome embodiments, X₁ is V. In some embodiments, X₂ is R. In someembodiments, X₂ is S. In some embodiments, X₂ is G. In some embodiments,X₃ is P. In some embodiments, X₃ is Y. In some embodiments, X₃ is R. Insome embodiments, X₃ is V. In some embodiments, X₃ is I. In someembodiments, X₃ is H. In some embodiments, X₃ is T. In some embodiments,X₃ is E. In some embodiments, X₄ is S. In some embodiments, X₄ is D. Insome embodiments, X₄ is E. In some embodiments, X₄ is N. In someembodiments, X₅ is D. In some embodiments, X₅ is A. In some embodiments,X₅ is T. In some embodiments, X₆ is S. In some embodiments, X₆ is G. Insome embodiments, X₆ is T. In some embodiments, X₆ is A. In someembodiments, X₇ is I. In some embodiments, X₇ is A. In some embodiments,X₇ is R. In some embodiments, X₇ is Q. In some embodiments, X₇ is V. Insome embodiments, X₈ is A. In some embodiments, X₈ is E. In someembodiments, X₈ is K. In some embodiments, X₈ is G. In some embodiments,X₈ is T. In some embodiments, X₉ is M. In some embodiments, X₉ is I. Insome embodiments, X₁ o is D. In some embodiments, X₁ o is L. In someembodiments, X₁ o is Q. In some embodiments, X₁ o is V. In someembodiments, X₁ o is N. In some embodiments, X₁ o is G. In someembodiments, X₁ i is V. In some embodiments, X₁ i is R. In someembodiments, X₁ i is N. In some embodiments, X₁ i is E. In someembodiments, X₁ i is K.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises three heavy chain CDRs and three light chain CDRs, wherein theheavy chain CDRs comprise:

-   -   a) CDR-H1 comprising the amino acid sequence        G(X₁)I(X₂)S(X₃)SYYW(X₄), wherein, optionally: X₁ is S or P; X₂        is S, H or R; X₃ is S or G; and, X₄ is G, I, N or V (SEQ ID NO:        395);    -   b) CDR-H2 comprising the amino acid sequence        SISYSA(X₁)TYYNPSLKS, wherein, optionally, X₁ is S or T (SEQ ID        NO: 396); and    -   c) CDR-H3 comprising the amino acid sequence

(X₁)(X₂)D(X₃)(X₄)Y(X₅)(X₆)(X₇)(X₈)G(X₉)(X₁₀)(X₁₁), wherein, optionally:X₁ is A or V; X₂ is R, S or G, X₃ is P, Y, R, V, I, H, T or E; X₄ is S,D, E or N; X₅ is D, A or T; X₆ is S, G, T or A; X₇ is I, A, R, Q, or V;X₈ is A, E, K, G or T; X₉ is M or I; X₁₀ is D, L, Q, V, N or G; and, X₁i is V, R, N, E or K (SEQ ID NO: 397).

In some embodiments, the antibody, or antigen-binding fragment thereof,further comprises a CDR-L1 as set forth in SEQ ID NO:103, optionallycomprising one or more amino acid changes. In some embodiment, theantibody, or antigen-binding fragment thereof, further comprises aCDR-L2 as set forth in SEQ ID NO:104, optionally comprising one or moreamino acid changes. In some embodiments, the antibody, orantigen-binding fragment thereof, further comprises a CDR-L3 as setforth in SEQ ID NO:105, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment,comprises at least three of the following six CDRs:

-   -   a) CDR-H1 comprising the amino acid sequence        G(X₁)I(X₂)S(X₃)SYYW(X₄), wherein, optionally: X₁ is S or P; X₂        is S, H or R; X₃ is S or G; and, X₄ is G, I, N or V (SEQ ID NO:        395);    -   b) CDR-H2 comprising the amino acid sequence        SISYSA(X₁)TYYNPSLKS, wherein, optionally, X₁ is S or T (SEQ ID        NO: 396);    -   c) CDR-H3 comprising the amino acid sequence        (X₁)(X₂)D(X₃)(X₄)Y(X₅)(X₆)(X₇)(X₈)G(X₉)(X₁o)(X₁₁), wherein,        optionally: X₁ is A or V; X₂ is R, S or G, X₃ is P, Y, R, V, I,        H, T or E; X₄ is S, D, E or N; X₅ is D, A or T; X₆ is S, G, T or        A; X₇ is I, A, R, Q, or V; X₈ is A, E, K, G or T; X₉ is M or I;        X₁₀ is D, L, Q, V, N or G; and, X₁ i is V, R, N, E or K (SEQ ID        NO: 397);    -   d) CDR-Ll: SEQ ID NO:103, optionally comprising one or more        amino acid changes;    -   e) CDR-L2: SEQ ID NO: 104, optionally comprising one or more        amino acid changes; and    -   f) CDR-L3: SEQ ID NO: 105, optionally comprising one or more        amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof,specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex.In some embodiments, the antibody, or antigen-binding fragment thereof,does not bind a human GARP-proTGFβ1 complex. In some embodiments, theantibody, or antigen-binding fragment thereof, does not bind matureTGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragment,specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex;wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or matureTGFβ33; wherein the antibody is a fully human or humanized antibody or afragment thereof, wherein the antibody comprises at least three of thefollowing six CDRs:

-   -   a) CDR-H1 comprising the amino acid sequence        G(X₁)I(X₂)S(X₃)SYYW(X₄), wherein, optionally: X₁ is S or P; X₂        is S, H or R; X₃ is S or G; and, X₄ is G, I, N or V (SEQ ID NO:        395);    -   b) CDR-H2 comprising the amino acid sequence        SISYSA(X₁)TYYNPSLKS, wherein, optionally, X₁ is S or T (SEQ ID        NO: 396);    -   c) CDR-H3 comprising the amino acid sequence        (X₁)(X₂)D(X₃)(X₄)Y(X₅)(X₆)(X₇)(X₈)G(X₉) (X₁₀)(X₁₁), wherein,        optionally: X₁ is A or V; X₂is R, S or G, X₃ is P, Y, R, V, I,        H, T or E; X₄ is S, D, E or N; X₅ is D, A or T; X₆ is S, G, T or        A; X₇ is I, A, R, Q, or V; X₈ is A, E, K, G or T; X₉ is M or I;        X₁₀ is D, L, Q, V, N or G; and, X₁ i is V, R, N, E or K (SEQ ID        NO: 397);    -   d) CDR-L1 as set forth in SEQ ID NO:103, optionally comprising        one or more amino acid changes;    -   e) CDR-L2 as set forth in SEQ ID NO: 104, optionally comprising        one or more amino acid changes; and    -   f) CDR-L3 as set forth in SEQ ID NO: 105, optionally comprising        one or more amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <100 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <100 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <50 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <50 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <25 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <25 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <10 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <10 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouseLTBP1-proTGFβ1. In some embodiments, such antibody is alsocross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <100 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <100 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI). In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <50 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <50 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI). In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <25 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <25 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI). In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <10 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <10 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to humanGARP-proTGFβ1. In preferred embodiments, such context-selective antibodyis also isoform-specific in that it selectively binds and inhibits theactivation of TGFβ1 associated with LTBP1/3.

In another aspect, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1 comprising the amino acid G(X₁)I(X₂)SSSYYW(X₃),wherein, optionally: X₁ is S or P; X₂ is H or R; and, X₃ is G, I or N(SEQ ID NO: 384). In some embodiments, X₁ is a S. In some embodiments,X₁ is a P. In some embodiments, X₂ is a H. In some embodiments, X₂ is anR. In some embodiments, X₃ is a G. In some embodiments, X₃ is an I. Insome embodiments, X₃ is an N.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H2 comprising the amino acid sequenceSISYSA(X₁)TYYNPSLKS, wherein, optionally, X₁ is S or T (SEQ ID NO: 396).In some embodiments, X₁ is a S. In some embodiment, X₁ is a T.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises a CDR-H3 comprising the amino acid sequenceA(X₁)D(X₂)SYD(X₃)(X₄)AGM(X₅)(X₆), wherein, optionally: X₁ is R, S or G,X₂ is P or V; X₃ is S or A; X₄ is A, R, I or V; X_(S) D, Q, or G; and,X₆ is V or R (SEQ ID NO: 385). In some embodiments, X₁ is R. In someembodiments, X₁ is S. In some embodiments, X₁ is G. In some embodiments,X₂ is P. In some embodiments, X₂ is V. In some embodiments, X₃ is S. Insome embodiments, X₃ is A. In some embodiments, X₄ is A. In someembodiments, X₄ is R. In some embodiments, X₄ is I. In some embodiments,X₄ is V. In some embodiments, X₅ is D. In some embodiments, X₅ is Q. Insome embodiments, X₅ is G. In some embodiments, X₆ is V. In someembodiments, X₆ is R.

In some embodiments, the antibody, or antigen-binding fragment thereof,comprises three heavy chain CDRs and three light chain CDRs, wherein theheavy chain CDRs comprise:

-   -   a) CDR-H1 comprising the amino acid sequence        G(X₁)I(X₂)SSSYYW(X₃), wherein, optionally: X₁ is S or P; X₂ is H        or R; and, X₃ is G, I or N (SEQ ID NO: 384);    -   b) CDR-H2 comprising the amino acid sequence        SISYSA(X₁)TYYNPSLKS, wherein, optionally, X₁ is S or T (SEQ ID        NO: 396); and    -   c) CDR-H3 comprising the amino acid sequence        A(X₁)D(X₂)SYD(X₃)(X₄)AGM(X₅)(X₆), wherein, optionally: X₁ is R,        S or G, X₂ is P or V; X₃ is S or A; X₄ is A, R, I or V; X₅ D, Q,        or G; and, X₆ is V or R (SEQ ID NO: 385).

In some embodiments, the antibody, or antigen-binding fragment thereof,further comprises a CDR-L1 as set forth in SEQ ID NO:103, optionallycomprising one or more amino acid changes. In some embodiment, theantibody, or antigen-binding fragment thereof, further comprises aCDR-L2 as set forth in SEQ ID NO:104, optionally comprising one or moreamino acid changes. In some embodiments, the antibody, orantigen-binding fragment thereof, further comprises a CDR-L3 as setforth in SEQ ID NO:105, optionally comprising one or more amino acidchanges.

In some embodiments, the antibody, or antigen-binding fragment,comprises at least three of the following six CDRs:

-   -   a) CDR-H1 comprising the amino acid sequence        G(X₁)I(X₂)SSSYYW(X₃), wherein, optionally: X₁ is S or P; X₂ is H        or R; and, X₃ is G, I or N (SEQ ID NO: 384);    -   b) CDR-H2 comprising the amino acid sequence        SISYSA(X₁)TYYNPSLKS, wherein, optionally, X₁ is S or T (SEQ ID        NO: 396);    -   c) CDR-H3 comprising the amino acid sequence        A(X₁)D(X₂)SYD(X₃)(X₄)AGM(X₅)(X₆), wherein, optionally: X₁ is R,        S or G, X₂ is P or V; X₃ is S or A; X₄ is A, R, I or V; X₅ D, Q,        or G; and, X₆ is V or R (SEQ ID NO: 385);    -   d) CDR-L1: SEQ ID NO:103, optionally comprising one or more        amino acid changes;    -   e) CDR-L2: SEQ ID NO: 104, optionally comprising one or more        amino acid changes; and    -   f) CDR-L3: SEQ ID NO: 105, optionally comprising one or more        amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof,specifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex.In some embodiments, the antibody, or antigen-binding fragment thereof,does not bind a human GARP-proTGFβ1 complex. In some embodiments, theantibody, or antigen-binding fragment thereof, does not bind matureTGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragmentthereof, specifically binds a human LTBP1-proTGFβ1 complex and/or ahuman LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1complex; wherein the antibody does not bind mature TGFβ1, mature TGFβ2or mature TGFβ33; wherein the antibody is a fully human or humanizedantibody or a fragment thereof, wherein the antibody comprises at leastthree of the following six CDRs:

-   -   a) CDR-H1 comprising the amino acid sequence        G(X₁)I(X₂)SSSYYW(X₃), wherein, optionally: X₁ is S or P; X₂ is H        or R; and, X₃ is G, I or N (SEQ ID NO: 384);    -   b) CDR-H2 comprising the amino acid sequence        SISYSA(X₁)TYYNPSLKS, wherein, optionally, X₁ is S or T (SEQ ID        NO: 396);    -   c) CDR-H3 comprising the amino acid sequence        A(X₁)D(X₂)SYD(X₃)(X₄)AGM(X₅)(X₆), wherein, optionally: X₁ is R,        S or G, X₂ is P or V; X₃ is S or A; X₄ is A, R, I or V; X₅ D, Q,        or G; and, X₆ is V or R (SEQ ID NO: 385);    -   d) CDR-L1: SEQ ID NO:103, optionally comprising one or more        amino acid changes;    -   e) CDR-L2: SEQ ID NO: 104, optionally comprising one or more        amino acid changes; and    -   f) CDR-L3: SEQ ID NO: 105, optionally comprising one or more        amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <100 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <100 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <50 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <50 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <25 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <25 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complexwith a KD of <10 nM as measured in a suitable in vitro binding assaysuch as Bio-Layer Interferometry (BLI); and/or, such antibody binds ahuman LTBP3-proTGFβ1 complex with a KD of <10 nM as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouseLTBP1-proTGFβ1. In some embodiments, such antibody is alsocross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <100 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <100 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI). In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <50 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <50 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI). In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <25 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <25 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI). In some embodiments, suchantibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <10 nM asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1complex with a KD of <10 nM as measured in a suitable in vitro bindingassay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to humanGARP-proTGFβ1. In preferred embodiments, such context-selective antibodyis also isoform-specific in that it selectively binds and inhibits theactivation of TGFβ1 associated with LTBP1/3.

Also provided herein are antibodies, or antigen-binding fragmentsthereof, which binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-TGFβ1 complex with yet higher affinities and further advantageouscombinations of binding properties.

Accordingly, in one aspect, the antibody, or antigen-binding fragmentthereof, comprises the following six CDRs:

-   -   a) CDR-H1 comprising the amino acid sequence FTFRSYVMH (SEQ ID        NO: 166);    -   b) CDR-H2 comprising the amino acid sequence        VISHEGS(X₁)KYYADSVKG, wherein: X₁ is L or G (SEQ ID NO: 366);        and    -   c) CDR-H3 comprising the amino acid sequence        A(X₁)PRIAARRGGFG(X₂), wherein: X₁ is V, R or L; and X₂ is Y, S        or T (SEQ ID NO: 367);    -   d) CDR-L1 comprising the amino acid sequence        TRS(X₁)G(X₂)ID(X₃)NYVQ, wherein, X₁ is S or H; X₂ is N, L, S or        A; and X₃ is N, D or Y (SEQ ID NO: 368);    -   e) CDR-L2 comprising the amino acid sequence ED(X₁)(X₂)RPS,        wherein: X₁ is N, F or A; and X₂ is Q, I or V (SEQ ID NO: 369);        and    -   f) CDR-L3 comprising the amino acid sequence        Q(X₁)YD(X₂)(X₃)(X₄)Q(X₅)VV, wherein: X₁ is S or G; X₂ is 5, F,        Y, D, H or W; X₃ is N, D or S; X₄ is N, A, L, E or T; and X₅ is        G, R, A or L (SEQ ID NO: 370).

In some embodiments, within CDR-H3: X₁ is R or L. Within CDR-L3: X₂ maybe Y. Within CDR-L3: X₃ may be D; and X₄ may be T. In some preferredembodiments, within CDR-H3: X₁ is R or L (optionally R), within CDR-L3:X₂ is Y; and within CDR-L3: X₃ is D; and X₄ is T.

In alternative embodiments, within CDR-H3: X₁ is R or L (optionally R),within CDR-L3: X₂ is Y and within CDR-L3: X₃ is D; X₄ is N; and X_(S) isA.

In some embodiments within CDR-L1: X₁ is S or H; X₂ is N or A; and X₃ isN, D or Y; within CDR-L2: X₁ is N or F; and X₂ is Q or V; and withinCDR-L3: X₁ is S or G; X₂ is 5, Y, D or W; X₃ is D or S; X₄ is N, L or T;and X₅ is G, R, A or L. In some embodiments, within CDR-L1: X₁ is S; X₂is N; and X₃ is N or Y; within CDR-L2: X₁ is N; and X₂ is Q or V; andwithin CDR-L3: X₁ is S or G; X₂ is 5, Y or W; X₃ is D; X₄ is N or T; andX₅ is G, R or A. In some embodiments, within CDR-L3: X₁ is S; X₂ is S orY; X₃ is D; X₄ is N or T; and X₅ is G, R or A. In some embodiments,within CDR-L3: X₁ is S; X₂ is Y; X₃ is D; X₄ is N or T; and X₅ is G orA. In some preferred embodiments, within CDR-L3: X₁ is S; X₂ is Y; X₃ isD; X₄ is T; and X₅ is G.

In particularly preferred embodiments, the antibody or antigen-bindingfragment has the CDRs of Ab42, e.g.: CDR-H1 comprising the amino acidsequence FTFRSYVMH (SEQ ID NO: 166); CDR-H2 comprising the amino acidsequence VISHEGSLKYYADSVKG (SEQ ID NO: 167); CDR-H3 comprising the aminoacid sequence ARPRIAARRGGFGY (SEQ ID NO: 168); CDR-L1 comprising theamino acid sequence TRSSGNIDNNYVQ (SEQ ID NO: 169); CDR-L2 comprisingthe amino acid sequence EDNQRPS (SEQ ID NO: 170); and CDR-L3 comprisingthe amino acid sequence QSYDYDTQGVV (SEQ ID NO: 171).

In some embodiments, the antibody or antigen-binding fragment comprisesa heavy chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 318; and a light chain variable regionhaving an amino acid sequence that is at least 90% identical to SEQ IDNO: 319.

In some embodiments, the antibody, or antigen-binding fragment, binds ahuman LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with aK_(D) of <5 nM as measured by suitable in vitro binding assay, such asBLI. For example, the antibody, or antigen-binding fragment, may bind ahuman LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with aK_(D) of <5 nM as measured by suitable in vitro binding assay, such asBLI. In some embodiments, the antibody, or antigen-binding fragment,binds the human LTBP1- and/or LTBP3-proTGFβ1 complex with a K_(D) of <1nM as measured by suitable in vitro binding assay, such as BLI.

In some embodiments, the antibody, or antigen-binding fragment thereofdoes not show detectable binding to a human GARP-proTGFβ1 complex, asmeasured by BLI, under the same assay conditions as used to measurebinding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1. Forexample, the antibody, or antigen-binding fragment may not showdetectable binding to a human GARP-proTGFβ1 complex, as measured by BLI,under the same assay conditions as used to measure binding to humanLTBP1-proTGFβ1 complex and human LTBP3-TGFβ1 complex.

In some embodiments, the antibody, or antigen-binding fragment thereof,binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complexwith a K_(D) that is at least 50 times lower (e.g., at least 75 timeslower, at least 100 times lower) than the K_(D) when binding to a humanGARP-proTGFβ1 complex under the same assay conditions. For example, theantibody, or antigen-binding fragment thereof, may bind a humanLTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K_(D) thatis at least 50 times lower (e.g., at least 75 times lower, at least 100times lower) than the K_(D) when binding to a human GARP-proTGFβ1complex under the same assay conditions. In some embodiments, K_(D) isas determined by BLI or SPR. In some embodiments, K_(D) is as determinedby SPR.

In some embodiments, the antibody, or antigen-binding fragment thereofdoes not show detectable binding to an LRRC33-proTGFβ1 latent complex,as measured by BLI, under the same assay conditions as used to measurebinding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1. Forexample, the antibody, or antigen-binding fragment thereof may not showdetectable binding to an LRRC33-proTGFβ1 latent complex, as measured byBLI, under the same assay conditions as used to measure binding to humanLTBP1-proTGFβ1 complex and human LTBP3-TGFβ1.

In some embodiments, the antibody, or antigen-binding fragment thereof,binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complexwith a K_(D) that is at least 50 times lower (e.g., at least 75 timeslower, at least 100 times lower) than the K_(D) when binding to a humanLRRC33-proTGFβ1 complex under the same assay conditions. For example,the antibody, or antigen-binding fragment thereof, may bind a humanLTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K_(D) thatis at least 50 times lower (e.g., at least 75 times lower, at least 100times lower) than the K_(D) when binding to a human LRRC33-proTGFβ1complex under the same assay conditions. In some embodiments, K_(D) isas determined by BLI or SPR. In some embodiments, K_(D) is as determinedby SPR.

In some embodiments, the antibody, or antigen-binding fragment thereof,is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, theantibody, or antigen-binding fragment thereof, is cross-reactive withmouse LTBP3-proTGFβ1. In some embodiments, the antibody, orantigen-binding fragment thereof, binds a mouse LTBP1-proTGFβ1 complexwith a K_(D) of <10 nM as measured by BLI. In some embodiments, theantibody, or antigen-binding fragment thereof, binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <10 nM as measured by BLI.

In further embodiments, the antibody which selectively binds a humanLTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex may not showmeaningful binding (e.g., may not show a response of more than 0.1 units(nm)) on exposure to a human GARP-proTGFβ1 complex in a BLI assay (e.g.,Octet) when the human GARP-proTGFβ1 complex is at a concentration of 200nM.

In another aspect, the antibody, or antigen-binding fragment thereof,comprises the following six CDRs:

-   -   a) CDR-H1 comprising the amino acid sequence        G(X₁)I(X₂)S(X₃)SYYW(X₄), wherein, optionally: X₁ is S; X₂ is S,        H or R; X₃ is S or G; and, X₄ is G, I, N or V (SEQ ID NO: 386);    -   b) CDR-H2 comprising the amino acid sequence SISYS(X₁)(X₂)TYY,        wherein, optionally: X₁ is G or A; and X₂ is S or T (SEQ ID NO:        398);    -   c) CDR-H3 comprising the amino acid sequence        A(X₁)DPSYDS(X₂)AGM(X₃)V, wherein, optionally: X₁ is R, S or G;        X₂ is A or I; and X₃ is D or Q (SEQ ID NO: 387);    -   d) CDR-L1 comprising the amino acid sequence        RAS(X₁)(X₂)IS(X₃)YLN, wherein, optionally: X₁ is K or Q; X₂ is V        or S; and X₃ is S or Y (SEQ ID NO: 389);    -   e) CDR-L2 comprising the amino acid sequence (X₁)AS(X₂)(X₃)QS,        wherein, optionally: X₁ is Y, A or S; X₂ is S or N; and X₃ is L        or R (SEQ ID NO: 390);    -   f) CDR-L3 comprising the amino acid sequence        QQ(X₁)(X₂)D(X₃)P(X₄)T, wherein, optionally: X₁ is S or G; X₂ is        F or N; X₃ is W or F; and X₄ is F or L (SEQ ID NO: 391).

In some embodiments: within CDR-H1: X₁ is S; X₂ is S or R; X₃ is 5; and,X₄ is G; within CDR-H2: X₁ is G or A; and X₂ is S or T; within CDR-H3:X₁ is R, S or G; X₂ is A or I; and X₃ is D or Q; within CDR-L1: X₁ is Kor Q; X₂ is V or S; and X₃ is S or Y; within CDR-L2: X₁ is Y, A or S; X₂is S or N; and X₃ is L or R; and within CDR-L3: X₁ is S or G; X₂ is F orN; X₃ is W or F; and X₄ is F or L.

In some embodiments: CDR-H1 comprises the amino acid sequenceGSIRSSSYYWG (SEQ ID NO: 292); CDR-H2 comprises the amino acid sequenceSISYSATTYY (SEQ ID NO: 293); within CDR-H3: X₁ is S or G; X₂ is A or I;and X₃ is D or Q; within CDR-L1: X₁ is K or Q; X₂ is V or S; and X₃ is Sor Y; within CDR-L2: X₁ is Y, A or S; X₂ is S or N; and X₃ is L or R;and within CDR-L3: X₁ is S or G; X₂ is F or N; X₃ is W or F; and X₄ is For L.

In some embodiments: CDR-H1 comprises the amino acid sequenceGSIRSSSYYWG (SEQ ID NO: 292); CDR-H2 comprises the amino acid sequenceSISYSATTYY (SEQ ID NO: 293); CDR-H3 comprises the amino acid sequenceAGDPSYDSIAGMQV (SEQ ID NO: 294); CDR-L1 comprises the amino acidsequence RASQSISSYLN (SEQ ID NO: 295); CDR-L2 comprises the amino acidsequence AASNLQS (SEQ ID NO: 296); and CDR-L3 comprises the amino acidsequence QQSFDWPLT (SEQ ID NO: 297).

In some embodiments, the antibody or antigen-binding fragment comprisesa heavy chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 360; and a light chain variable regionhaving an amino acid sequence that is at least 90% identical to SEQ IDNO: 361.

In some embodiments, the antibody, or antigen-binding fragment, binds ahuman LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with aK_(D) of <5 nM as measured by suitable in vitro binding assay, such asBLI. For example, the antibody, or antigen-binding fragment, may bind ahuman LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with aK_(D) of <5 nM as measured by suitable in vitro binding assay, such asBLI. In some embodiments, the antibody, or antigen-binding fragment,binds the human LTBP1- and/or LTBP3-proTGFβ1 complex with a K_(D) of <1nM as measured by suitable in vitro binding assay, such as BLI.

In some embodiments, the antibody, or antigen-binding fragment thereofdoes not show detectable binding to a human GARP-proTGFβ1 complex, asmeasured by BLI, under the same assay conditions as used to measurebinding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1. Forexample, the antibody, or antigen-binding fragment may not showdetectable binding to a human GARP-proTGFβ1 complex, as measured by BLI,under the same assay conditions as used to measure binding to humanLTBP1-proTGFβ1 complex and human LTBP3-TGFβ1 complex.

In some embodiments, the antibody, or antigen-binding fragment thereof,binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complexwith a K_(D) that is at least 50 times lower (e.g., at least 75 timeslower, at least 100 times lower) than the K_(D) when binding to a humanGARP-proTGFβ1 complex under the same assay conditions. For example, theantibody, or antigen-binding fragment thereof, may bind a humanLTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K_(D) thatis at least 50 times lower (e.g., at least 75 times lower, at least 100times lower) than the K_(D) when binding to a human GARP-proTGFβ1complex under the same assay conditions. In some embodiments, K_(D) isas determined by BLI or SPR. In some embodiments, K_(D) is as determinedby SPR.

In some embodiments, the antibody, or antigen-binding fragment thereofdoes not show detectable binding to an LRRC33-proTGFβ1 latent complex,as measured by BLI, under the same assay conditions as used to measurebinding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1. Forexample, the antibody, or antigen-binding fragment thereof may not showdetectable binding to an LRRC33-proTGFβ1 latent complex, as measured byBLI, under the same assay conditions as used to measure binding to humanLTBP1-proTGFβ1 complex and human LTBP3-TGFβ1.

In some embodiments, the antibody, or antigen-binding fragment thereof,binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complexwith a K_(D) that is at least 50 times lower (e.g., at least 75 timeslower, at least 100 times lower) than the K_(D) when binding to a humanLRRC33-proTGFβ1 complex under the same assay conditions. For example,the antibody, or antigen-binding fragment thereof, may bind a humanLTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K_(D) thatis at least 50 times lower (e.g., at least 75 times lower, at least 100times lower) than the K_(D) when binding to a human LRRC33-proTGFβ1complex under the same assay conditions. In some embodiments, K_(D) isas determined by BLI or SPR. In some embodiments, K_(D) is as determinedby SPR.

In some embodiments, the antibody, or antigen-binding fragment thereof,is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, theantibody, or antigen-binding fragment thereof, is cross-reactive withmouse LTBP3-proTGFβ1. In some embodiments, the antibody, orantigen-binding fragment thereof, binds a mouse LTBP1-proTGFβ1 complexwith a K_(D) of <10 nM as measured by BLI. In some embodiments, theantibody, or antigen-binding fragment thereof, binds a mouseLTBP3-proTGFβ1 complex with a K_(D) of <10 nM as measured by BLI.

In further embodiments, the antibody which selectively binds a humanLTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex may not showmeaningful binding (e.g., may not show a response of more than 0.1 units(nm)) on exposure to a human GARP-proTGFβ1 complex in a BLI assay (e.g.,Octet) when the human GARP-proTGFβ1 complex is at a concentration of 200nM.

In some embodiments, the “percent identity” of two amino acid sequencesis determined using the algorithm of Karlin and Altschul Proc. Natl.Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and AltschulProc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm isincorporated into the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searchescan be performed with the XBLAST program, score=50, word length=3 toobtain amino acid sequences homologous to the protein molecules ofinterest. Where gaps exist between two sequences, Gapped BLAST can beutilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs,the default parameters of the respective programs (e.g., XBLAST andNBLAST) can be used.

In any of the antibodies or antigen-binding fragments described herein,one or more conservative mutations can be introduced into the CDRs orframework sequences at positions where the residues are not likely to beinvolved in an antibody-antigen interaction. In some embodiments, suchconservative mutation(s) can be introduced into the CDRs or frameworksequences at position(s) where the residues are not likely to beinvolved in interacting with a LTBP1-TGFβ complex and/or a LTBP3-TGFβcomplex, as determined based on the crystal structure. In someembodiments, the likely interface (e.g., residues involved in anantigen-antibody interaction) may be deduced from known structuralinformation on another antigens sharing structural similarities.

As used herein, a “conservative amino acid substitution” refers to anamino acid substitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g., MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989,or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the antibodies provided herein comprise mutationsthat confer desirable properties to the antibodies. For example, toavoid potential complications due to Fab-arm exchange, which is known tooccur with native IgG4 mAbs, the antibodies provided herein may comprisea stabilizing ‘Adair’ mutation (Angal et al., “A single amino acidsubstitution abolishes the heterogeneity of chimeric mouse/human (IgG4)antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EUnumbering; residue 241 Kabat numbering) is converted to prolineresulting in an IgG1-like (CPPCP (SEQ ID NO: 45)) hinge sequence.Accordingly, any of the antibodies may include a stabilizing ‘Adair’mutation or the amino acid sequence CPPCP (SEQ ID NO: 45). In oneembodiment, an antibody described herein comprises a heavy chainimmunoglobulin constant domain of a human IgG₄ having a backbonesubstitution of Ser to Pro, that produces an IgG₁-like hinge and permitsformation of inter-chain disulfide bonds.

Antibodies of this disclosure that selectively bind to a LTBP1-TGFβcomplex and/or a LTBP3-TGFβ complex may optionally comprise antibodyconstant regions or parts thereof. For example, a V_(L) domain may beattached at its C-terminal end to a light chain constant domain such asCκ or Cλ. Similarly, a V_(H) domain or portion thereof may be attachedto all or part of a heavy chain such as IgA, IgD, IgE, IgG, and IgM, andany isotype subclass. Antibodies may include suitable constant regions(see, for example, Kabat et al., Sequences of Proteins of ImmunologicalInterest, No. 91-3242, National Institutes of Health Publications,Bethesda, Md. (1991)). Therefore, antibodies within the scope of thismay disclosure include V_(H) and V_(L) domains, or antigen-bindingportions thereof, combined with any suitable constant region. Inexemplary embodiments, the antibodies, or antigen-binding portionsthereof, comprise a heavy chain immunoglobulin constant domaincontaining all or a portion of a human IgGi or a human IgG₄ constantdomain In some embodiments, the antibody, or antigen-binding portionthereof, comprises a light chain immunoglobulin constant domaincontaining all or a portion of a human Ig lambda constant domain or ahuman Ig kappa constant domain.

In some embodiments, antibodies that selectively bind to a LTBP1-TGFβcomplex and/or a LTBP3-TGFβ complex may or may not include the frameworkregion of the antibodies of SEQ ID NOs: 7 and 8. In some embodiments,antibodies that selectively bind to a LTBP1-TGFβ complex and/or aLTBP3-TGFβ complex are murine antibodies and include murine frameworkregion sequences. In other embodiments, the antibodies are chimericantibodies, or antigen-binding fragments thereof. In another embodiment,the antibodies are humanized antibodies, or antigen-binding fragmentsthereof. In another embodiment, the antibodies are fully humanantibodies, or antigen-binding fragments thereof. In one embodiment, theantibody comprises a framework region comprising a human germline aminoacid sequence.

The antibodies, and antigen-binding fragments thereof, described hereincan have any configuration suitable for binding antigen. For example, inone embodiment, the antibody, or antigen-binding portion thereof,comprises four polypeptide chains, including two heavy chain variableregions and two light chain variable regions. In another embodiment, theantibody, or antigen-binding portion thereof, comprises one heavy chainvariable region and one light chain variable region. In exemplaryembodiments, the antibody, or antigen-binding portion thereof, is a Fabfragment, a F(ab′)2 fragment, a scFab fragment, an scFv, or a diabody.

In one embodiment, the antibody, or antigen-binding portion thereof,comprises a heavy chain immunoglobulin constant domain of a human IgGiconstant domain or a human IgG₄ constant domain In an exemplaryembodiment, the heavy chain immunoglobulin constant domain is a humanIgG₄ constant domain In one embodiment, the antibody, or antigen-bindingportion thereof, binds a conformational epitope. In one embodiment, theantibody, or antigen-binding portion thereof, binds a combinatorialepitope.

In one embodiment, the antibody, or antigen-binding portion thereof,comprises a heavy chain immunoglobulin constant domain of a human IgG₄constant domain having a backbone substitution of Ser to Pro thatproduces an IgG₁-like hinge and permits formation of inter-chaindisulfide bonds. In one embodiment, the antibody, or antigen-bindingportion thereof, further comprises a light chain immunoglobulin constantdomain comprising a human Ig lambda constant domain, or a human Ig kappaconstant domain.

In one embodiment, the antibody is an IgG having four polypeptide chainswhich are two heavy chains and two light chains. In exemplaryembodiments, the antibody can be a humanized antibody, a human antibody,or a chimeric antibody. In one embodiment, the antibody comprises aframework having a human germline amino acid sequence.

In one embodiment, the invention provides an antibody, orantigen-binding portion thereof, that competes for binding with anantibody, or antigen-binding portion thereof, described herein. In oneembodiment, the invention provides an antibody, or antigen-bindingportion thereof, that binds to the same epitope as an antibody, orantigen-binding portion thereof, described herein. In one embodiment,the antibody, or antigen-binding fragment thereof, does not compete withantibody SR-Ab1 for binding to a human LTBP1-proTGFβ1 complex.

Binding Kinetics of Novel Antibodies

The novel antibodies and antigen-binding fragments thereof (e.g., Fabs)disclosed herein are characterized by enhanced binding properties. Theantibodies and the fragments are capable of selectively binding to aLTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex (also known as largelatent complexes (LLCs). Recombinantly produced, purified proteincomplexes may be used as antigens (e.g., antigen complexes) to screen,evaluate or confirm the ability of an antibody to bind the antigencomplexes in suitable in vitro binding assays. Such assays are wellknown in the art and include but are not limited to: BLI-based assays(such as Octet®) and SPR-based assays (such as Biacore).

Previously, antibodies and fragments that exhibited high affinities(e.g., sub-nanomolar K_(D)) to the LLCs were identified. Here,advantageously, antibodies and fragments with particularly slowdissociation rates were specifically selected, aimed to achieveparticularly durable inhibitory effects.

Accordingly, selection of suitable TGFβ inhibitors for carrying out themethods and therapeutic use in accordance with the present disclosuremay include carrying out in vitro binding assays to measure bindingkinetics. In preferred embodiments, the antibody or the antigen-bindingfragment binds hLTBP1-pro TGFβ1 and/or hLTBP3-proTGFβ1 with highaffinity and low dissociation rate k_(OFF), as described herein.Preferably, the antibody or the fragment further binds the murine LLCcounterparts, namely, mLTBP1-proTGFβ1 and/or mLTBP3-proTGFβ1, withequivalent affinities as the human LLCs. In vitro binding kinetics maybe readily determined by measuring interactions of test antibodies (suchas antigen-binding fragments) and suitable antigen, such as LLCs andsmall latent complexes (SLCs). Suitable methods for in vitro bindingassays to determine the parameters of binding kinetics include BLI-basedassays such as Octet, and surface plasmon resonance-based assays, suchas Biacore systems. An example of an Octet-based in vitro binding assayis provided in Example 9/Table 9. Several antibodies were shown in thisexperiment to have “OFF” rates (k_(OFF)) that are <5×10⁻⁴ (1/s). Theseresults are in stark contrast to the results shown for Ab10, for whichbinding to hLTBP1-pro TGFβ1 and/or hLTBP3-proTGFβ1 could not even bedetected by the same Octet assay (Table 8). An example of SPR-based invitro binding assay is provided in Example 9. Fab fragments of Ab42,Ab63 and Ab43, which are activation inhibitors of TGFβ1, were used inthis experiment. As illustrated in this example, these antibodies havesub-nanomolar K_(D) and “OFF” rates that are <5×10⁻⁴ (1/s). Thus, forexample, Ab42 is able to remain bound to the antigen for a much longerduration of time (e.g., greater t½) than an antibody with much higher“OFF” rate, which “falls off” (e.g., dissociates from) the antigenrelatively quickly. Thus, the difference in the dissociation kineticspredominantly attributes to the notable difference in their overallaffinities (K_(D)), which may result in enhanced potency. Therefore,characterization of binding kinetics provides useful information as topotential durability of effects and resulting in vivo potency.

Accordingly, the invention includes a method of selecting a TGFβactivation inhibitor for therapeutic use, wherein the method comprisesselection of an antibody or antigen-binding fragment thereof that has adissociation rate of <5×10⁻⁴ ((1/s) as measured by SPR. In someembodiments, the antibody or the fragment binds antigen with an affinityof less than 1 nM (i.e., sub-nanomolar), e.g., less than 500 pM, 400 pM,300 pM, 200 pM, 100 pM, or 50 pM.

The selection method may include determining or measuring dissociationhalf-time (also referred to as half binding time or t′) of test antibodyby suitable means, such as BLI-based assays and SPR-based assays.Monovalent or multivalent (e.g., divalent) test antibodies may be used.For example, Fab fragments are suitable monovalent antibodies that canbe used to determine dissociation half time. Similarly, full lengthimmunoglobulins (e.g.,IgGs) may be used to determine dissociation halftime. In some embodiments, the method comprises selection of an antibodyor antigen-binding fragment thereof that has t′ of 45 minutes or longerto human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1. Preferably, theantibody or the fragment has t′ of 45 minutes or longer to each of humanLTBP1-proTGFβ1 and human LTBP3-proTGFβ1. More preferably, the antibodyor the fragment further has t′ of 45 minutes or longer to murineLTBP1-proTGFβ1 and/or murine LTBP3-proTGFβ1. Most preferably, theantibody or the fragment has t′ of 45 minutes or longer to each ofmurine LTBP1-proTGFβ1 and murine LTBP3-proTGFβ1. The method may furtherinclude selection of an antibody or an antigen-binding fragment that hast½ of 5 minutes or less to human GARP-proTGFβ1. Any of these antibodieswith advantagenous dissociation half time should preferably have a KD ofless than 1 nM as measured by BLI (e.g., Octet) or SPR (e.g., Biacore).Preferably, SPR assays are used to determine dissociation half-time(t½).

Polypeptides

Some aspects of the disclosure relate to isolated polypeptides. Forexample, in one embodiment, the invention provides an isolatedpolypeptide comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, orcombinations thereof, as provided in Table 5. In an exemplaryembodiment, the isolated polypeptide can contain CDRH1, CDRH2, and CDRH3as provided in Table 5. In other embodiments, the isolated polypeptidecan contain CDRL1, CDRL2, and CDRL3 as provided in Table 5. In someembodiments, the polypeptide can contain up to 6, 5, 4, 3, 2, or 1 aminoacid residue variations as compared to the corresponding CDR region inany one of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinationsthereof, as provided in Table 5. In one embodiment, the inventionprovides an isolated polypeptide comprising SEQ ID NO: 7. In anotherembodiment, the invention provides an isolated polypeptide comprisingSEQ ID NO: 8. In another embodiment, the invention provides an isolatedpolypeptide comprising SEQ ID NO:7 and SEQ ID NO:8. In this embodiment,SEQ ID NO:7 and SEQ ID NO:8 can optionally be connected by a linkerpeptide. In some embodiments, the polypeptide is a heavy chain variabledomain. In some embodiments, the polypeptide is at least 75% (e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 7. In someembodiments, the polypeptide is a light chain variable domain. In someembodiments, the polypeptide is at least 75% (e.g., 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%) identical to SEQ ID NO: 8.

In another embodiment, the invention provides an isolated polypeptidecomprising a heavy chain variable region sequence set forth in Table 6.In one embodiment, the invention provides an isolated polypeptidecomprising SEQ ID NO: 7, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, or SEQ ID NO: 106. In one embodiment, the inventionprovides an isolated polypeptide comprising SEQ ID NO: 8, SEQ ID NO:75,SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85,SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, or SEQ ID NO:107. In one embodiment, the invention provides an isolated polypeptidecomprising SEQ ID NO: 318. In one embodiment, the invention provides anisolated polypeptide comprising SEQ ID NO: 319. In one embodiment, theinvention provides an isolated polypeptide comprising SEQ ID NO: 360. Inone embodiment, the invention provides an isolated polypeptidecomprising SEQ ID NO: 361.

In another embodiment, the invention provides an isolated polypeptidecomprising a light chain variable region set forth in Table 6. Inanother embodiment, the invention provides an isolated polypeptidecomprising a heavy chain variable region sequence set forth in Table 6(e.g., SEQ ID NO: 7, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, or SEQ ID NO: 106) and a light chain variableregion sequence set forth in Table 6 (e.g., SEQ ID NO: 8, SEQ ID NO:75,SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85,SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, or SEQ ID NO:107). In this embodiment, the heavy chain and light chain sequences(e.g., SEQ ID NO:7 and SEQ ID NO:8) can optionally be connected by alinker peptide. In some embodiments, the polypeptide is a heavy chainvariable domain In some embodiments, the polypeptide is at least 75%(e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 7, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, or SEQ IDNO: 106. In some embodiments, the polypeptide is a light chain variabledomain. In some embodiments, the polypeptide is at least 75% (e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 8, SEQ ID NO:75, SEQID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, or SEQ ID NO: 107.

In another embodiment, the invention provides an isolated polypeptidecomprising a heavy chain variable region sequence set forth in SEQ IDNO: 318 and a light chain variable region sequence set forth in SEQ IDNO: 319. In another embodiment, the invention provides an isolatedpolypeptide comprising a heavy chain variable region sequence set forthin SEQ ID NO: 360 and a light chain variable region sequence set forthin SEQ ID NO: 361. In this embodiment, the heavy chain and light chainsequences (e.g., SEQ ID NO: 318 and SEQ ID NO: 319) can be connected bya linker peptide. In some embodiments, the polypeptide is a heavy chainvariable domain In some embodiments, the polypeptide is at least 85%(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical toSEQ ID NO: 318 or SEQ ID NO: 360. In some embodiments, the polypeptideis a light chain variable domain. In some embodiments, the polypeptideis at least 85% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99%) identical to SEQ ID NO: 319 or SEQ ID NO: 361.

Nucleic Acids

In some embodiments, antibodies, antigen-binding portions thereof,and/or compositions of the present disclosure may be encoded by nucleicacid molecules. Such nucleic acid molecules include, without limitation,DNA molecules, RNA molecules, polynucleotides, oligonucleotides, mRNAmolecules, vectors, plasmids and the like. In some embodiments, thepresent disclosure may comprise cells programmed or generated to expressnucleic acid molecules encoding compounds and/or compositions of thepresent disclosure.

In some embodiments, the invention provides a nucleic acid molecule thatencodes the foregoing antibodies, or an antigen-binding portion thereof.For example, in one embodiment, the invention provides a nucleic acidmolecule that encodes a polypeptide comprising CDRH1, CDRH2, CDRH3,CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided in Table 5.The nucleic acid molecule can, in some embodiments, encode a polypeptidecomprising CDRH1, CDRH2, and CDRH3 as provided in Table 5. In someembodiments, the nucleic acid molecule can encode a polypeptidecomprising CDRL1, CDRL2, and CDRL3 as provided in Table 5. In someembodiments, the nucleic acid molecule encodes a polypeptide that cancontain up to 6, 5, 4, 3, 2, or 1 amino acid residue variations ascompared to the corresponding CDR region in any one of CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided inTable 5. In an exemplary embodiment, the nucleic acid molecule encodes apolypeptide comprising a heavy chain variable domain having at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to theamino acid sequence set forth in SEQ ID NO: 7, and/or a light chainvariable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:8. In one embodiment, the nucleic acid molecule encodes a polypeptidecomprising a heavy chain variable domain having at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a heavy chain variableregion sequence set forth in Table 6, and/or a light chain variabledomain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identity to a light chain variable region sequence set forth inTable 6. In an exemplary embodiment, the nucleic acid molecule encodes apolypeptide comprising a heavy chain variable domain having at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to theamino acid sequence set forth in SEQ ID NO: 7, and/or a light chainvariable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the nucleic acid molecule encodes an antibody,or antigen-binding portion thereof, comprising a heavy chain variabledomain amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO:74, SEQID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, or SEQ ID NO: 106, anda light chain variable domain amino acid sequence set forth in SEQ IDNO: 8, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ IDNO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ IDNO:93, or SEQ ID NO: 107. In some embodiments, the nucleic acid moleculeencodes an antibody, or antigen-binding portion thereof, comprising aheavy chain variable domain amino acid sequence set forth in SEQ ID NO:7, and a light chain variable domain amino acid sequence set forth inSEQ ID NO: 8.

In another embodiment, the invention provides nucleic acid molecule thatencodes the foregoing antibodies, or an antigen-binding portion thereof.In an exemplary embodiment, the nucleic acid molecule encodes apolypeptide comprising a heavy chain variable domain having at least85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequenceset forth in SEQ ID NO: 318, and/or a light chain variable domain havingat least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acidsequence set forth in SEQ ID NO: 319. In annother embodiment, thenucleic acid molecule encodes a polypeptide comprising a heavy chainvariable domain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence set forth in SEQ ID NO: 360, and/ora light chain variable domain having at least 85%, 90%, 95%, 96%, 97%,98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:361.

In another embodiment, the invention provides a nucleic acid moleculethat encodes a polypeptide comprising a heavy chain variable regionsequence set forth in SEQ ID NO: 318 and a light chain variable regionsequence set forth in SEQ ID NO: 319. In another embodiment, theinvention provides a nucleic acid molecule that encodes an isolatedpolypeptide comprising a heavy chain variable region sequence set forthin SEQ ID NO: 360 and a light chain variable region sequence set forthin SEQ ID NO: 361. In this embodiment, the heavy chain and light chainsequences (e.g., SEQ ID NO: 318 and SEQ ID NO: 319) can be connected bya linker peptide. In some embodiments, the nucleic acid molecule encodesa polypeptide which is a heavy chain variable domain In someembodiments, the nucleic acid molecule encodes a polypeptide which is atleast 85% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)identical to SEQ ID NO: 318 or SEQ ID NO: 360. In some embodiments, thenucleic acid molecule encodes a polypeptide which is a light chainvariable domain In some embodiments, the nucleic acid molecule encodes apolypeptide which is at least 85% (e.g., 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%) identical to SEQ ID NO: 319 or SEQ ID NO: 361. Insome embodiments, the nucleic acid molecule encodes an antibody, orantigen-binding portion thereof, comprising a heavy chain variabledomain amino acid sequence set forth in SEQ ID NO: 318, and a lightchain variable domain amino acid sequence set forth in SEQ ID NO: 319.In some embodiments, the nucleic acid molecule encodes an antibody, orantigen-binding portion thereof, comprising a heavy chain variabledomain amino acid sequence set forth in SEQ ID NO: 360, and a lightchain variable domain amino acid sequence set forth in SEQ ID NO: 361.

In some cases, nucleic acids of the disclosure include codon-optimizednucleic acids. Methods of generating codon-optimized nucleic acids areknown in the art and may include, but are not limited to those describedin US Patent Nos. 5,786,464 and 6,114,148, the contents of each of whichare herein incorporated by reference in their entirety. Also providedherein are expression vectors comprising any of the aforementionednucleus acid(s).

Production of Antibodies that Bind a LTBP1/3-TGFβ1 Complex

The art is familiar with various techniques and methods that may be usedfor obtaining antibodies, or antigen-binding fragments thereof, of thedisclosure. For example, antibodies can be produced using recombinantDNA methods, hybridoma techniques, phage or yeast display technologies,transgenic animals (e.g., a XenoMouse®) or some combination thereof.

Immunization and Hybridomas

In some methods described herein, the specified antigen (e.g., anLTBP1-TGFβ1 complex and/or an LTBP3-TGFβ1 complex) can be used toimmunize a non-human animal (“host”), e.g., a rodent, e.g., a mouse,hamster, or rat. In one embodiment, the non-human animal is a mouse. Inanother embodiment, the host may be a camelid.

After immunization, which may include single or multiple steps ofantigen exposures (e.g., injections), splenocytes are harvested from theanimal and the associated B cells are fused with immortalized myelomacells to form hybridomas for antibody production. Hybridomas may begenerated in accordance with known methods (see e.g., Kohler andMilstein (1975) Nature, 256: 495-499). Hybridomas formed in this mannerare then screened using standard methods, such as enzyme-linkedimmunosorbent assay (ELISA), Bio-Layer Interferometry (BLI) technology(e.g., OCTET) and surface plasmon resonance (e.g., BIACORE) analysis, toidentify one or more hybridomas that produce an antibody thatspecifically binds to a specified antigen. Any form of the specifiedantigen may be used as the immunogen, e.g., recombinant antigen,naturally occurring forms, any variants or fragments thereof, as well asantigenic peptide thereof (e.g., any of the epitopes described herein asa linear epitope or within a scaffold as a conformational epitope).

Screening Library/Libraries

In some embodiments, the method or process of making or identifyingantibodies includes a step of screening protein expression librariesthat express antibodies or fragments thereof (e.g., scFv), e.g., phage,yeast, or ribosome display libraries. For example, a library of humancombinatorial antibodies or scFv fragments can synthesized on phages oryeast, the library is then screened with the antigen of interest or anantibody-binding portion thereof, and the phage or yeast that binds theantigen is isolated, from which one may obtain the antibodies orimmunoreactive fragments (Vaughan et a/., 1996, PMID: 9630891; Sheets eta/., 1998, PMID: 9600934; Boder et a/., 1997, PMID: 9181578; Pepper eta/., 2008, PMID: 18336206).

Phage display is further described, for example, in Ladner et al., U.S.Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al.(1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO93/01288; WO 92/01047; WO 92/09690; and WO 90/02809. Yeast display isfurther described, for example, in U.S. Pat. Nos. 7,700,302 and8,877,688. In particular methods, the yeast display library expressesfull-length antibodies (e.g., Adimab, LLC).

Kits for generating phage or yeast display libraries are commerciallyavailable. There also are other methods and reagents that can be used ingenerating and screening antibody display libraries (see US 5,223,409;WO 92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO92/01047, WO 92/09690; and Barbas et a/., 1991, PMID: 1896445). Suchtechniques advantageously allow for the screening of large numbers ofcandidate antibodies.

No matter how obtained, antibody producing cells (e.g., yeast colonies,hybridomas, etc.) may be selected, cloned and further screened fordesirable characteristics including, for example, robust growth, highantibody production and desirable antibody characteristics such as highaffinity for the antigen of interest. Methods of selecting, cloning andexpanding colonies and/or hybridomas are well known to those of ordinaryskill in the art. Once the desired antibodies are identified, therelevant genetic material may be isolated, manipulated, and expressedusing common, art-recognized molecular biology and biochemicaltechniques.

Humanization

The antibodies or fragments of the present invention are preferablyfully human antibodies or humanized antibodies. Thus, whatever thesource, it will be appreciated that the method may comprise humanizingone or more antibodies or fragments thereof, wherein the human antibodysequences may be fabricated using art-known molecular engineeringtechniques and introduced into expression systems and host cells asdescribed herein. Such non-natural recombinantly produced humanantibodies (and subject compositions) are entirely compatible with theteachings of this disclosure and are expressly held to be within thescope of the instant invention. In certain select aspects, theLTBP1-TGFβ1 complex-binding and/or a LTBP3-TGFβ1 complex-bindingantibodies of the invention will comprise a recombinantly produced humanantibody.

Affinity maturation

In some embodiments, antibodies produced by the methods described-abovemay be of moderate affinity (Ka of about 10⁶ to 10⁷M-1). Accordingly,antibodies or fragments thereof may be subjected to a process ofaffinity maturation as part of optimization, if desired. The term“affinity maturation” shall have the meaning readily understood by theskilled artisan. Briefly, it refers to further modifying the amino acidsequence of candidate antibodies or fragments (often referred to as“parent”) to achieve improved binding profiles to the specific antigen.Typically, a parental antibody and an affinity-matured counterpart(sometimes referred to as “progeny” or “offspring”) retain the sameepitope. Suitable in vitro binding assays may be carried out to screenfor improved binders at appropriate step(s) during the affinitymaturation process. Optionally, in some embodiments, functional assays(e.g., cell-based potency assays) may also be performed to confirmdesired functionality.

Affinity maturation typically involves sequence diversification and/ormutagenesis, whilst the exact means of introducing or generatingmutations is not limiting. In some embodiments, mutagenesis comprisesintroducing one or more changes (e.g., substitutions or deletions) inamino acid residues of one or more CDRs. Accordingly, in someembodiments the VR or CDR sequences described herein may comprise up toone, two, three, four, five, or six amino acid changes. In someembodiments, the VR or CDR sequences described herein may comprise up toone, two, three, four, five, or six amino acid substitutions. In someembodiments, the VR or CDR sequences described herein may comprise up toone, two, three, or four deletions. Additionally or alternatively,mutagenesis may comprise so-called oligo-walking of variable regions orCDRs.

For example, affinity maturation of antibodies can be accomplished by anumber of methods including random mutagenesis (Gram H.,et al. Proc.Natl. Acad. Sci. U.S.A. (1992) 89, 3576-3580, and Hawkins R. E., et al.J. Mol. Biol. (1992) 226, 889-896), random mutagenesis of CDR sequences,e.g., CDR walking (Yang W. P., et al., J. Mol. Biol. (1995) 254,392-403), directed mutagenesis of residues (Ho M., et al., J. Biol.Chem. (2005) 280, 607-617 and Ho M., et al., Proc. Natl. Acad. Sci.U.S.A. (2006) 103, 9637-9642), and approaches that reproduce somatichypermutation (SHM) in vitro (Bowers P. M., et al., Proc. Natl. Acad.Sci. U.S.A. (2011) 108, 20455-20460).

In one embodiment, antibodies may be affinity matured by conductingmutagenesis on the variable heavy or variable light chain regions. Inanother embodiment, antibodies may be affinity matured by conductingmutagenesis on any one of the variable heavy chain CDRs or variablelight chain CDRs. In another embodiment, antibodies may be affinitymatured by conducting mutagenesis on the variable heavy chain CDR3(e.g., CDR-H3 mutagenesis). In another embodiment, antibodies may beaffinity matured by conducting mutagenesis on the variable heavy chainCDR2 (e.g., CDR-H2 mutagenesis). In another embodiment, antibodies maybe affinity matured by conducting mutagenesis on the variable heavychain CDR1 (e.g., CDR-H1 mutagenesis). In another embodiment, antibodiesmay be affinity matured by conducting mutagenesis on the variable lightchain CDR3 (e.g., CDR-L3 mutagenesis). In another embodiment, antibodiesmay be affinity matured by conducting mutagenesis on the variable lightchain CDR2 (e.g., CDR-L2 mutagenesis). In another embodiment, antibodiesmay be affinity matured by conducting mutagenesis on the variable lightchain CDR1 (e.g., CDR-L1 mutagenesis).

In some embodiments, an antibody may be affinity matured and/oroptimized by separately conducting mutagenesis on some or all of thethree light chain CDRs (i.e., CDR-L1, CDR-L2, and CDR-L3) to generate upto three different libraries of antibodies, each having unique mutationsin one of the three CDRs. The new antibodies can then be screened forimproved properties (e.g., antigen-binding). Then, if further affinityimprovements are desired, unique CDRs from each of the libraries can bemixed and matched to generate a new library of antibodies and screenedfor improved properties (e.g., antigen-binding). In some embodiments,affinity maturation involves screening an antibody library comprisingvariants of one or more CDRs (“repertoire”), which may be combined withat least one CDR of a parent antibody. This process is sometimes calledCDR shuffling or CDR diversification. In some embodiments, a variableheavy chain CDR3 (e.g., CDR-H3) may be used to screen a library thatcontains variable heavy chain CDR1 and CDR2 repertoires of variants(a.k.a., CDR-H1/H2 diversification). In some embodiments, variable lightchain CDR3 (e.g., CDR-L3) may be used to screen a library that containsvariable light chain CDR1 and CDR2 repertoires of variants (a.k.a.,CDR-L1/L2 diversification).

In some embodiments, affinity maturation involves screening an antibodylibrary comprising light chain variants (“repertoire”), which may becombined with a heavy chain of a parent antibody (light-chainshuffling). For example, in some embodiments, selected heavy chains areintroduced into an antibody library comprising light chain variantsthereby producing a new library of antibodies that can be screened forimproved affinity. In some embodiments, repertoires of naturallyoccurring variable region variants may be obtained from unimmunizeddonors. Examples of heavy or light-chain shuffling are described in thefollowing documents: Marks et al., (1992) Nature Biotech 10: 779-78;Schier et al., (1996) J. Mol. Biol. 255, 28-43; Park et al., (2000)BBRC. 275. 553-557; and Chames et al., (2002) J. Immunol 1110-1118. Insome embodiments, the light chain library comprises lambda light chainsvariants. In some embodiments, the light chain library comprises kappalight chains variants. In some embodiments, the light chain librarycomprises both lambda and kappa light chains variants.

It should be appreciated that the various methods for affinitymaturation may be combined in any order. For example, in one embodiment,a select antibody may undergo heavy chain CDR-H1/H2 diversification,followed by CDR-H3 mutagenesis. In another embodiment, a select antibodymay undergo heavy chain CDR-H1/H2 diversification, followed by CDR-H3mutagenesis, followed by light chain shuffling. In another embodiment, aselect antibody may undergo heavy chain CDR-H1/H2 diversification,followed by light chain shuffling, followed by CDR-H3 mutagenesis. Inanother embodiment, a select antibody may undergo light chain shuffling,followed by heavy chain CDR-H1/H2 diversification, followed by CDR-H3mutagenesis. In another embodiment, a select antibody may undergo lightchain shuffling, followed by heavy chain CDR-H1/H2 diversification,followed by CDR-H3 mutagenesis, followed by CDR-L3 mutagenesis.

In some embodiment, a select antibody may undergo light chain shuffling,followed by heavy chain CDR-H1/H2 diversification, followed by CDR-H3mutagenesis, followed by CDR-L1, CDR-L2, and/or CDRL3 mutagenesis. Insome embodiments, a select antibody may undergo heavy chain CDR-H1/H2diversification, followed by CDR-H3 mutagenesis, followed by CDR-L1,CDR-L2, and/or CDRL3 mutagenesis. In some embodiments, light chain CDRmutagenesis is separately conducted on some or all of the three lightchain CDRs (CDR-L1, CDR-L2, and CDRL3) to produce up to three librariesof antibodies. In some embodiments, the light chain CDRs from each ofthe libraries are mixed and matched to generate a new library ofantibodies having unique combinations of light chain CDR mutations.

In any of the methods for affinity maturation described above, theresulting new antibodies may be selected for binding to the targetantigen (e.g., a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex) usingknown techniques (e.g., FACS). Binding specificity and affinity usingFACS may be tested by varying antigen concentration and/or competitionfor unlabeled (cold) antigen. Binding affinity can be further assessedusing other techniques known in the art, such as ELISA, BLI (e.g.,OCTET), and SPR (e.g., BIACORE).

In addition to the affinity maturation process discussed above, furtheroptimization may be performed to achieve desired product profiles. Thus,antibodies may be further subjected to a step of optimization andselected based on certain physicochemical properties that areadvantageous. For therapeutic antibodies (biologics), physicochemicalcriteria for developability that may be evaluated include, but are notlimited to: solubility, stability, immunogenicity, lack ofself-association or aggregation, Fc functionality, internalizationprofiles, pH-sensitivity, glycosylation and manufacturability such ascell viability and/or gene expression. In some embodiments, the processof optimization involves mutagenesis of one or more amino acid sequenceswithin the constant regions.

In one aspect, the invention provides a method for making a compositioncomprising an antibody, or antigen-binding fragment thereof, thatspecifically binds a human LTBP1-proTGFβ1 complex and/or a humanLTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex;wherein the antibody, or antigen-binding fragment thereof, inhibitsTGFβ1 but does not inhibit TGFβ2 or TGFβ3, the method comprising stepsof i) providing at least one antigen comprising LTBP1-proTGFβ1 and/orLTBP3-proTGFβ31, ii) selecting a first pool of antibodies, orantigen-binding fragments thereof, that specifically bind the at leastone antigen of step (i) so as to provide specific binders ofLTBP1-proTGFβ1 and/or LTBP3-proTGFβ31; iii) selecting a second pool ofantibodies, or antigen-binding fragments thereof, that inhibitactivation of TGFβ1, so as to generate specific inhibitors of TGFβ1activation; iv) formulating an antibody, or antigen-binding fragmentthereof, that is present in the first pool of antibodies and the secondpool of antibodies into a pharmaceutical composition, thereby making thecomposition comprising the antibody, or antigen-binding fragmentthereof.

In one embodiment, the method further comprises a step of removing fromthe first pool of antibodies, or antigen-binding fragments thereof, anyantibodies, or antigen-binding fragments thereof, that bindGARP-proTGFβ1, LRRC33-proTGFβ1, mature TGFβ1, GARP-proTGFβ2,LRRC33-proTGFβ2, mature TGFβ2, GARP-proTGFβ3, LRRC33-proTGFβ3, matureTGFβ3, or any combinations thereof. In one embodiment, the methodfurther comprises a step of determining or confirmingisoform-specificity of the antibodies, or antigen-binding fragmentsthereof, selected in steps (ii) and/or (iii). In one embodiment, themethod further comprises a step of selecting for antibodies, orantigen-binding fragments thereof, that are cross-reactive to human androdent antigens. In one embodiment, the method further comprises a stepof generating a fully human or humanized antibody, or antigen-bindingfragment thereof, of the antibody, or antigen-binding fragment thereof,that is present in the first pool of antibodies and the second pool ofantibodies.

In one embodiment, the method further comprises a step of subjecting theantibody, or antigen-binding fragment thereof, that is present in thefirst pool of antibodies and/or the second pool of antibodies toaffinity maturation and/or optimization, so as to provide an affinitymatured and/or optimized antibody or fragment thereof. In oneembodiment, the affinity maturation and/or optimization comprises a stepof subjecting the antibody, or antigen-binding fragment thereof, tolight chain shuffling as described herein. In one embodiment, theaffinity maturation and/or optimization comprises the step of subjectingthe antibody, or antigen-binding fragment thereof, to CDR H1/H2diversification as described herein. In one embodiment, the affinitymaturation and/or optimization comprises the step of subjecting theantibody, or antigen-binding fragment thereof, to CDR-H3 mutagenesis asdescribed herein. In one embodiment, the affinity maturation and/oroptimization comprises the step of subjecting antibody, orantigen-binding fragment thereof, to light chain CDR mutagenesis asdescribed herein. In one embodiment, the affinity maturation and/oroptimization comprises the step of subjecting the antibody, orantigen-binding fragment thereof, to light chain CDR L1/L2diversification as described herein.

Further optimization steps may be carried out to provide physicochemicalproperties that are advantageous for therapeutic compositions. Suchsteps may include, but are not limited to, mutagenesis or engineerringto provide improved solubility, lack of self-aggregation, stability, pHsensitivity, Fc function, and so on.

In one embodiment, the method further comprises a step of determiningaffinity of the antibodies, or antigen-binding fragments thereof, tohuman LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1. In some embodiments,the method further comprises a step of removing from the first and/orsecond pools of antibodies, or antigen-binding fragments thereof, anyantibodies, or antigen-binding fragments thereof, that bind to humanLTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K_(D) of >100 nM, asmeasured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI). In some embodiments, the method further comprisesa step of removing from the first and/or second pools of antibodies, orantigen-binding fragments thereof, any antibodies, or antigen-bindingfragments thereof, that bind to human LTBP1-proTGFβ1 and/or humanLTBP3-proTGFβ1 with a K_(D) of >50 nM, as measured in a suitable invitro binding assay such as Bio-Layer Interferometry (BLI). In someembodiments, the method further comprises a step of removing from thefirst and/or second pools of antibodies, or antigen-binding fragmentsthereof, any antibodies, or antigen-binding fragments thereof, that bindto human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K_(D) of >25nM, as measured in a suitable in vitro binding assay such as Bio-LayerInterferometry (BLI). In some embodiments, the method further comprisesa step of removing from the first and/or second pools of antibodies, orantigen-binding fragments thereof, any antibodies, or antigen-bindingfragments thereof, that bind to human LTBP1-proTGFβ1 and/or humanLTBP3-proTGFβ1 with a K_(D) of >10 nM, as measured in a suitable invitro binding assay such as Bio-Layer Interferometry (BLI).

In one embodiment, the method further comprises a step of determiningaffinity of the antibodies, or antigen-binding fragments thereof, fromthe first and/or second pools of antibodies to mouse LTBP1-proTGFβ1and/or mouse LTBP3-proTGFβ1. In some embodiments, the method furthercomprises a step of removing from the first and/or second pools ofantibodies, or antigen-binding fragments thereof, any antibodies, orantigen-binding fragments thereof, that bind to mouse LTBP1-proTGFβ1and/or mouse LTBP3-proTGFβ1 with a K_(D) of >100 nM, as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, the method further comprises a step of removingfrom the first and/or second pools of antibodies, or antigen-bindingfragments thereof, any antibodies, or antigen-binding fragments thereof,that bind to mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with aK_(D) of >50 nM, as measured in a suitable in vitro binding assay suchas Bio-Layer Interferometry (BLI). In some embodiments, the methodfurther comprises a step of removing from the first and/or second poolsof antibodies, or antigen-binding fragments thereof, any antibodies, orantigen-binding fragments thereof, that bind to mouse LTBP1-proTGFβ1and/or mouse LTBP3-proTGFβ1 with a K_(D) of >25 nM, as measured in asuitable in vitro binding assay such as Bio-Layer Interferometry (BLI).In some embodiments, the method further comprises a step of removingfrom the first and/or second pools of antibodies, or antigen-bindingfragments thereof, any antibodies, or antigen-binding fragments thereof,that bind to mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with aK_(D) of >10 nM, as measured in a suitable in vitro binding assay suchas Bio-Layer Interferometry (BLI).

In one embodiment, the method further comprises a step of removing fromthe first and/or second pool of antibodies, or antigen-binding fragmentsthereof, any antibodies, or antigen-binding fragments thereof, that donot bind mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1.

In one embodiment, the method further comprises a step of determiningthe IC₅₀ of the antibodies, or antigen-binding fragments thereof, fromthe first and/or second pools of antibodies, or antigen-bindingfragments thereof, as measured by a suitable functional in vitrocell-based assay such as a caga assay, as described herein. In someembodiments, the method comprises the step of removing antibodies, orantigen-binding fragments thereof, from the first and/or second pools ofantibodies, or antigen-binding fragments thereof, that have an IC₅₀ ofgreater than 50 nM as measured by a caga assay as described herein. Insome embodiments, the method comprises the step of removing antibodies,or antigen-binding fragments thereof, from the first and/or second poolsof antibodies, or antigen-binding fragments thereof, that have an IC₅₀of greater than 25 nM as measured by a caga assay as described herein.In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof, from the first and/orsecond pools of antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 10 nM as measured by a caga assay asdescribed herein. In some embodiments, the method comprises the step ofremoving antibodies, or antigen-binding fragments thereof, from thefirst and/or second pools of antibodies, or antigen-binding fragmentsthereof, that have an IC₅₀ of greater than 5 nM as measured by a cagaassay as described herein.

In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof, from the first and/orsecond pools of antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 100 nM as measured by an endogenous LTBPcaga assay as described herein. In some embodiments, the methodcomprises the step of removing antibodies, or antigen-binding fragmentsthereof, from the first and/or second pools of antibodies, orantigen-binding fragments thereof, that have an IC₅₀ of greater than 50nM as measured by an endogenous LTBP caga assay as described herein. Insome embodiments, the method comprises the step of removing antibodies,or antigen-binding fragments thereof, from the first and/or second poolsof antibodies, or antigen-binding fragments thereof, that have an IC₅₀of greater than 25 nM as measured by an endogenous LTBP caga assay asdescribed herein. In some embodiments, the method comprises the step ofremoving antibodies, or antigen-binding fragments thereof, from thefirst and/or second pools of antibodies, or antigen-binding fragmentsthereof, that have an IC₅₀ of greater than 10 nM as measured by anendogenous LTBP caga assay as described herein. In some embodiments, themethod comprises the step of removing antibodies, or antigen-bindingfragments thereof, from the first and/or second pools of antibodies, orantigen-binding fragments thereof, that have an IC₅₀ of greater than 5nM as measured by an endogenous LTBP caga assay as described herein.

In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof, from the first and/orsecond pools of antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 100 nM as measured by a human LTBPoverexpression caga assay as described herein. In some embodiments, themethod comprises the step of removing antibodies, or antigen-bindingfragments thereof, from the first and/or second pools of antibodies, orantigen-binding fragments thereof, that have an IC₅₀ of greater than 50nM as measured by a human LTBP overexpression caga assay as describedherein. In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof, from first and/orsecond pools of antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 25 nM as measured by a human LTBPoverexpression caga assay as described herein. In some embodiments, themethod comprises the step of removing antibodies, or antigen-bindingfragments thereof, from the first and/or second pools of antibodies, orantigen-binding fragments thereof, that have an IC₅₀ of greater than 10nM as measured by a human LTBP overexpression caga assay as describedherein. In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof, from the first and/orsecond pools of antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 5 nM as measured by a human LTBPoverexpression caga assay as described herein.

In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof, from the first and/orsecond pools of antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 100 nM as measured by a murine LTBPoverexpression caga assay as described herein. In some embodiments, themethod comprises the step of removing antibodies, or antigen-bindingfragments thereof, from the first and/or second pools of antibodies, orantigen-binding fragments thereof, that have an IC₅₀ of greater than 50nM as measured by a murine LTBP overexpression caga assay as describedherein. In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof, from first and/orsecond pools of antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 25 nM as measured by a murine LTBPoverexpression caga assay as described herein. In some embodiments, themethod comprises the step of removing antibodies, or antigen-bindingfragments thereof, from first and/or second pools of antibodies, orantigen-binding fragments thereof, that have an IC₅₀ of greater than 10nM as measured by a murine LTBP overexpression caga assay as describedherein. In some embodiments, the method comprises the step of removingantibodies, or antigen-binding fragments thereof from first and/orsecond pools of antibodies, or antigen-binding fragments thereof, thathave an IC₅₀ of greater than 5 nM as measured by a murine LTBPoverexpression caga assay as described herein.

In another embodiment, a monoclonal antibody is obtained from thenon-human animal, and then modified, e.g., made chimeric, using suitablerecombinant DNA techniques. A variety of approaches for making chimericantibodies have been described. See e.g., Morrison et al., Proc. Natl.Acad. Sci. U.S.A. 81:6851, 1985; Takeda et al., Nature 314:452, 1985,Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No.4,816,397; Tanaguchi et al., European Patent Publication EP171496;European Patent Publication 0173494, United Kingdom Patent GB 2177096B.

For additional antibody production techniques, see, e.g., Antibodies: ALaboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory,1988. The present disclosure is not necessarily limited to anyparticular source, method of production, or other specialcharacteristics of an antibody.

Some aspects of the present disclosure relate to host cells transformedwith a polynucleotide or vector. Host cells may be a prokaryotic oreukaryotic cell. The polynucleotide or vector which is present in thehost cell may either be integrated into the genome of the host cell orit may be maintained extrachromosomally. The host cell can be anyprokaryotic or eukaryotic cell, such as a bacterial, insect, fungal,plant, animal or human cell. In some embodiments, fungal cells are, forexample, those of the genus Saccharomyces, in particular those of thespecies S. cerevisiae. The term “prokaryotic” includes all bacteriawhich can be transformed or transfected with a DNA or RNA molecules forthe expression of an antibody or the corresponding immunoglobulinchains. Prokaryotic hosts may include gram negative as well as grampositive bacteria such as, for example, E. coli, S. typhimurium,Serratia marcescens and Bacillus subtilis. The term “eukaryotic”includes yeast, higher plants, insects and vertebrate cells, e.g.,mammalian cells, such as NSO and CHO cells. Depending upon the hostemployed in a recombinant production procedure, the antibodies orimmunoglobulin chains encoded by the polynucleotide may be glycosylatedor may be non-glycosylated. Antibodies or the correspondingimmunoglobulin chains may also include an initial methionine amino acidresidue.

In some embodiments, once a vector has been incorporated into anappropriate host, the host may be maintained under conditions suitablefor high level expression of the nucleotide sequences, and, as desired,the collection and purification of the immunoglobulin light chains,heavy chains, light/heavy chain dimers or intact antibodies,antigen-binding fragments or other immunoglobulin forms may follow; see,Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y.,(1979). Thus, polynucleotides or vectors are introduced into the cellswhich in turn produce the antibody or antigen-binding fragments.Furthermore, transgenic animals, preferably mammals, comprising theaforementioned host cells may be used for the large scale production ofthe antibody or antibody fragments.

The transformed host cells can be grown in fermenters and cultured usingany suitable techniques to achieve optimal cell growth. Once expressed,the whole antibodies, their dimers, individual light and heavy chains,other immunoglobulin forms, or antigen-binding fragments, can bepurified according to standard procedures of the art, including ammoniumsulfate precipitation, affinity columns, column chromatography, gelelectrophoresis and the like; see, Scopes, “Protein Purification”,Springer Verlag, N.Y. (1982). The antibody or antigen-binding fragmentscan then be isolated from the growth medium, cellular lysates, orcellular membrane fractions. The isolation and purification of the,e.g., microbially expressed antibodies or antigen-binding fragments maybe by any conventional means such as, for example, preparativechromatographic separations and immunological separations such as thoseinvolving the use of monoclonal or polyclonal antibodies directed, e.g.,against the constant region of the antibody.

Aspects of the disclosure relate to a hybridoma, which provides anindefinitely prolonged source of monoclonal antibodies. As analternative to obtaining immunoglobulins directly from the culture ofhybridomas, immortalized hybridoma cells can be used as a source ofrearranged heavy chain and light chain loci for subsequent expressionand/or genetic manipulation. Rearranged antibody genes can be reversetranscribed from appropriate mRNAs to produce cDNA. In some embodiments,heavy chain constant region can be exchanged for that of a differentisotype or eliminated altogether. The variable regions can be linked toencode single chain Fv regions. Multiple Fv regions can be linked toconfer binding ability to more than one target or chimeric heavy andlight chain combinations can be employed. Any appropriate method may beused for cloning of antibody variable regions and generation ofrecombinant antibodies.

In some embodiments, an appropriate nucleic acid that encodes variableregions of a heavy and/or light chain is obtained and inserted into anexpression vectors which can be transfected into standard recombinanthost cells. A variety of such host cells may be used. In someembodiments, mammalian host cells may be advantageous for efficientprocessing and production. Typical mammalian cell lines useful for thispurpose include CHO cells, 293 cells, or NSO cells. The production ofthe antibody or antigen-binding fragment may be undertaken by culturinga modified recombinant host under culture conditions appropriate for thegrowth of the host cells and the expression of the coding sequences. Theantibodies or antigen-binding fragments may be recovered by isolatingthem from the culture. The expression systems may be designed to includesignal peptides so that the resulting antibodies are secreted into themedium; however, intracellular production is also possible.

The disclosure also includes a polynucleotide encoding at least avariable region of an immunoglobulin chain of the antibodies describedherein. In some embodiments, the variable region encoded by thepolynucleotide comprises at least one complementarity determining region(CDR) of the VH and/or VL of the variable region of the antibodyproduced by any one of the above described hybridomas.

Polynucleotides encoding antibody or antigen-binding fragments may be,e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or arecombinantly produced chimeric nucleic acid molecule comprising any ofthose polynucleotides either alone or in combination. In someembodiments, a polynucleotide is part of a vector. Such vectors maycomprise further genes such as marker genes which allow for the β thevector in a suitable host cell and under suitable conditions.

In some embodiments, a polynucleotide is operatively linked toexpression control sequences allowing expression in prokaryotic oreukaryotic cells. Expression of the polynucleotide comprisestranscription of the polynucleotide into a translatable mRNA. Regulatoryelements ensuring expression in eukaryotic cells, preferably mammaliancells, are well known to those skilled in the art. They may includeregulatory sequences that facilitate initiation of transcription andoptionally poly-A signals that facilitate termination of transcriptionand stabilization of the transcript. Additional regulatory elements mayinclude transcriptional as well as translational enhancers, and/ornaturally associated or heterologous promoter regions. Possibleregulatory elements permitting expression in prokaryotic host cellsinclude, e.g., the PL, Lac, Trp or Tac promoter in E. coli, and examplesof regulatory elements permitting expression in eukaryotic host cellsare the AOX₁ or GAL1 promoter in yeast or the CMV-promoter,SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer,SV40-enhancer or a globin intron in mammalian and other animal cells.

Beside elements which are responsible for the initiation oftranscription such regulatory elements may also include transcriptiontermination signals, such as the SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. Furthermore, depending on theexpression system employed, leader sequences capable of directing thepolypeptide to a cellular compartment or secreting it into the mediummay be added to the coding sequence of the polynucleotide and have beendescribed previously. The leader sequence(s) is (are) assembled inappropriate phase with translation, initiation and terminationsequences, and preferably, a leader sequence capable of directingsecretion of translated protein, or a portion thereof, into, forexample, the extracellular medium. Optionally, a heterologouspolynucleotide sequence can be used that encode a fusion proteinincluding a C- or N-terminal identification peptide imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product.

In some embodiments, polynucleotides encoding at least the variabledomain of the light and/or heavy chain may encode the variable domainsof both immunoglobulin chains or only one. Likewise, polynucleotides maybe under the control of the same promoter or may be separatelycontrolled for expression. Furthermore, some aspects relate to vectors,particularly plasmids, cosmids, viruses and bacteriophages usedconventionally in genetic engineering that comprise a polynucleotideencoding a variable domain of an immunoglobulin chain of an antibody orantigen-binding fragment; optionally in combination with apolynucleotide that encodes the variable domain of the otherimmunoglobulin chain of the antibody.

In some embodiments, expression control sequences are provided aseukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells, but control sequences forprokaryotic hosts may also be used. Expression vectors derived fromviruses such as retroviruses, vaccinia virus, adeno-associated virus,herpes viruses, or bovine papilloma virus, may be used for delivery ofthe polynucleotides or vector into targeted cell population (e.g., toengineer a cell to express an antibody or antigen-binding fragment). Avariety of appropriate methods can be used to construct recombinantviral vectors. In some embodiments, polynucleotides and vectors can bereconstituted into liposomes for delivery to target cells. The vectorscontaining the polynucleotides (e.g., the heavy and/or light variabledomain(s) of the immunoglobulin chains encoding sequences and expressioncontrol sequences) can be transferred into the host cell by suitablemethods, which vary depending on the type of cellular host.

Modifications

Antibodies, or antigen-binding portions thereof, of the disclosure maybe modified with a detectable label or detectable moiety, including, butnot limited to, an enzyme, prosthetic group, fluorescent material,luminescent material, bioluminescent material, radioactive material,positron emitting metal, nonradioactive paramagnetic metal ion, andaffinity label for detection and/or isolation of a LTBP1-TGFβ1 complexor a LTBP3-TGFβ1 complex. The detectable substance or moiety may becoupled or conjugated either directly to the polypeptides of thedisclosure or indirectly, through an intermediate (such as, for example,a linker (e.g., a cleavable linker)) using suitable techniques.Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, oracetylcholinesterase; non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin; non-limitingexamples of suitable fluorescent materials include biotin,umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin;an example of a luminescent material includes luminol; non-limitingexamples of bioluminescent materials include luciferase, luciferin, andaequorin; and examples of suitable radioactive material include aradioactive metal ion, e.g., alpha-emitters or other radioisotopes suchas, for example, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I) carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹¹⁵mIn, ¹¹³mIn, ¹¹²In, ¹¹¹In), andtechnetium (⁹⁹Tc, ⁹⁹mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd) molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, 166Ho, ⁹⁰Y, ⁴⁷Sc, ⁸⁶R, ¹⁸⁸Re,¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr,⁵⁴Mn, ⁷⁵Se, and tin (¹¹³Sn, ¹¹⁷Sn). The detectable substance may becoupled or conjugated either directly to the antibodies of thedisclosure that bind selectively to a LTBP1-TGFβ1 complex and/or aLTBP3-TGFβ1 complex, or indirectly, through an intermediate (such as,for example, a linker) using suitable techniques. Any of the antibodiesprovided herein that are conjugated to a detectable substance may beused for any suitable diagnostic assays, such as those described herein.

In addition, antibodies, or antigen-binding portions thereof, of thedisclosure may also be modified with a drug to form, e.g., anantibody-drug conjugate. The drug may be coupled or conjugated eitherdirectly to the polypeptides of the disclosure, or indirectly, throughan intermediate (such as, for example, a linker (e.g., a cleavablelinker)) using suitable techniques.

Targeting Agents

In some embodiments methods of the present disclosure comprise the useof one or more targeting agents to target an antibody, orantigen-binding portion thereof, as disclosed herein, to a particularsite in a subject for purposes of enriching or localizing such agent(s)to a niche of interest. In some embodiments, such targeting may achievemodulating mature TGFβ release from a LTBP1-TGFβ1 complex and/or aLTBP3-TGFβ1 complex. For example, LTBP1-TGFβ1 and LTBP3-TGFβ1 complexesare typically localized to extracellular matrix. Thus, in someembodiments, antibodies disclosed herein can be conjugated toextracellular matrix targeting agents for purposes of localizing theantibodies to sites where LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes reside.In such embodiments, selective targeting of antibodies leads toselective modulation of LTBP1-TGFβ1 and/or LTBP3-TGFβ1 complexes. Insome embodiments, selective targeting of antibodies leads to selectiveinhibition of LTBP1-TGFβ1 and/or LTBP3-TGFβ1 complexes (e.g., forpurposes of treating fibrosis). In some embodiments, extracellularmatrix targeting agents include heparin binding agents, matrixmetalloproteinase binding agents, lysyl oxidase binding domains,fibrillin-binding agents, hyaluronic acid binding agents, and others.

In some embodiments, bispecific antibodies may be used having a firstportion that selectively binds a LTBP1/3-TGFβ1 complex and a secondportion that selectively binds a component of a target site, e.g., acomponent of the ECM (e.g., fibrillin).

In some embodiments, such a target agent may be coupled to another agentor therapeutics to carry or localize the complex to a niche of interest.

Safety/Toxicity Considerations Histopathology, Toxicology

As mentioned above, known pan-inhibitors that antagonize all TGFβisoforms, namely, TGFβ1, TGFβ2 and TGFβ3, have been documented to causevarious toxicities across multiple mammalian species. Most notable knowntoxicities include cardiovascular toxicities (such as valvulopathy) andepithelial hyperplasia, skin lesions, inflammation and bleeding. Morespecifically, some of the observed toxicities associated with pan-TGFβinhibitors (e g , small molecule antagonists of the TGFβR andnon-selective neutralizing antibodies) reported in the literatureinclude the following.

Cardiovascular toxicities associated with TGFβ inhibition include,hyperplasia in aortic valve, right AV valve, and left AV valve;inflammation in aortic valve, left AV valve, and ascending aorta;hemorrhage in ascending aorta, aortic valve and left AV valve;connective tissue degeneration in ascending aorta (see for example,Strauber et al. (2014) “Nonclinical safety evaluation of a TransformingGrowth Factor _(R) receptor I kinase inhibitor in Fischer 344 rats andbeagle dogs” J. Clin. Pract 4(3): 1000196).

In addition, neutralizing antibodies that bind all three TGFβ isoformshave been associated with certain epithelial toxicities, which aresummarized in the table below.

Epithelial and other toxicities across species for 1D11 and GC1008 MiceCyno Human Toxicities Hyperplasia and Hyperplasia of Gingival bleedinginflammation of gingiva, nasal Epistaxis tongue, gingiva, epithelium,Headache and esophagus. and bladder Fatigue Findings not Anemia lead toVarious skin reversible cessation of disorders, including (12 wkrecovery) treatment keratoacanthomas Changes were (KA), hyperkeratosis,reversible (except cutaneous SCC, and bladder) basal cell carcinomaDrug/ 1D11 GC1008 GC1008 Dose/ Dosing: 50 Dosing: 10 and Dose: 0.1, 0.3,Duration mg/kg(3×/week) 50 mg/kg 1, 3, 10, 15 mg/kg Duration: Duration:Duration: 4 9-12 weeks 6 months monthly doses Exposure Serum conc. = Notdisclosed Half-life: 21.7 d 1-2 mg/mL DN Cmax~(350 (over 4-12 weeks)ng/mL)mg *Vitsky et. Al. Am. J Pathology vol. 174, 2009; and Lonning et.al. Current Pharmaceutical Biotech, 2011

Applicant of the present disclosure previously demonstrated the improvedsafety profiles of monoclonal antibodies that selectively block theactivation step of TGFβ1 by targeting latent proTGFβ1 complex (see, forexample, WO 2017/156500 and WO 2018/129329). In rat toxicology studiesdescribed therein, there were no observable test article-relatedtoxicities when the animals were dosed with the inhibitors up to 100mg/kg per week for 4 weeks.

Building upon the earlier recognition by the applicant of the presentdisclosure (see PCT/US2017/021972) that lack of isoform-specificity ofconventional TGFβ antagonists may underlie the source of toxicitiesassociated with TGFβ inhibition, the present inventors sought to furtherachieve context-selective TGFβ1 inhibition for treating various diseasesthat manifest TGFβ1 dysregulation, particularly fibrotic conditions,with enhanced safety/tolerability. The work presented herein thereforefurther provided, among high-affinity inhibitors, a subset of antibodieswith particularly low dissociation rates (k_(OFF)) in order to improvedurability.

Thus, in some embodiments, the novel antibody according to the presentdisclosure has the maximally tolerated dose (MTD) of >100 mg/kg whendosed weekly for at least 4 weeks (e.g., 4, 6, 8, 10, 12 weeks). In someembodiments, the novel antibody according to the present disclosure hasthe no observed adverse effect level (NOAEL) of up to 100 mg/kg whendosed weekly for at least 4 weeks in rats. In some embodiments, theantibody has a NOAEL of at least 100 mg/kg/week when dosed for 4 weeksor 12 weeks in mice. Suitable animal models to be used for conductingsafety/toxicology studies for TGFβ inhibitors and TGFβ1 inhibitorsinclude, but are not limited to: rats, dogs, cynos, and mice. Inpreferred embodiments, the minimum effective amount of the antibodybased on a suitable preclinical efficacy study is below the NOAEL. Morepreferably, the minimum effective amount of the antibody is aboutone-third or less of the NOAEL. In particularly preferred embodiments,the minimum effective amount of the antibody is about one-sixth or lessof the NOAEL. In some embodiments, the minimum effective amount of theantibody is about one-tenth or less of the NOAEL.

In some embodiments, the invention encompasses an isoform-selectiveantibody capable of inhibiting TGFβ1 signaling, which, when administeredto a subject, does not cause cardiovascular or known epithelialtoxicities at a dose effective to treat a TGFβ1-related indication. Insome embodiments, the antibody has a minimum effective amount of about3-10 mg/kg administered weekly, biweekly or monthly. Preferably, theantibody causes no to minimum toxicities at a dose that is at leastsix-times the minimum effective amount (e.g., a six-fold therapeuticwindow). More preferably, the antibody causes no to minimum toxicitiesat a dose that is at least ten-times the minimum effective amount (e.g.,a ten-fold therapeutic window). Even more preferably, the antibodycauses no to minimum toxicities at a dose that is at least fifteen-timesthe minimum effective amount (e.g., a fifteen-fold therapeutic window).

Thus, selection of an antibody or an antigen-binding fragment thereoffor therapeutic use may include: selecting an antibody orantigen-binding fragment that meets the criteria of one or more of TGFβinhibitors (such as monoclonal antibodies and antigen-binding fragmentsselected for example for having slow dissociation rates, e.g., k_(OFF)of <5×10 (1/s)); carrying out an in vivo efficacy study in a suitablepreclinical model to determine an effective amount of the antibody orthe fragment; carrying out an in vivo safety/toxicology study in asuitable model to determine an amount of the antibody that is safe ortoxic (e.g., MTD, NOAEL, or any art-recognized parameters for evaluatingsafety/toxicity); and, selecting the antibody or the fragment thatprovides at least a three-fold therapeutic window (preferably 6-fold,more preferably a 10-fold therapeutic window, even more preferably a15-fold therapeutic window). The selected antibody or the fragment maybe used in the manufacture of a pharmaceutical composition comprisingthe antibody or the fragment. Such pharmaceutical composition may beused in the treatment of a TGFβ1 indication in a subject as describedherein. For example, the TGFβ1 indication may be a fibrotic disorder.Preferably, a TGFβ3 inhibitor to be selected for therapeutic use orlarge-scale manufacture, does not produce observable adverse effects inthe treated animals after at least 4 week, e.g., 8 weeks, and 12 weeks,of sustained exposure. In some embodiments, certain toxicities observedin histopathological analyses are considered non-adverse.

Immune Safety Assessment

Cytokines play an important role in normal immune responses, but whenthe immune system is triggered to become hyperactive, the positivefeedback loop of cytokine production can lead to a “cytokine storm” orhypercytokinemia, a situation in which excessive cytokine productioncauses an immune response that can damage organs, especially the lungsand kidneys, and even lead to death. Such condition is characterized bymarkedly elevated proinflammatory cytokines in the serum. Historically,a Phase 1 Trial of the anti-CD28 monoclonal antibody TGN1412 in healthyvolunteers led to a life-threatening “cytokine storm” response resultedfrom an unexpected systemic and rapid induction of proinflammatorycytokines (Suntharalingam G et al. N Engl J Med. 2006 Sep 7;355(10):1018-28). This incident prompted heightened awareness of thepotential danger associated with pharmacologic stimulation of T cells.

Whilst TGF13-directed therapies do not target a specific T cell receptoror its ligand, it is contemplated that it is prudent to carry out immunesafety assessment, including, for example, in vitro cytokine releaseassays, in vivo cytokine measurements from plasma samples of non-humanprimate treated with a TGFβ3 inhibitor, and platelet assays using humanplatelets.

In some embodiments, selection of a TGFβ3 inhibitor for therapeutic useand/or large-scale production thereof includes an assessment of theability for the TGFβ3 inhibitor to trigger cytokine release fromcytokine-producing cells. In such an assessment, one or more of thecytokines (e.g., inflammatory cytokines) IL-2, TNFα, IFNγ, IL-1β, CCL2(MCP-1), and IL-6 may be assayed. In some embodiments, thecytokine-producing cells may include peripheral blood mononuclear cell(PBMC) constituents from heathy donors. Cytokine response after exposureto the TGFβ3 inhibitor (such as an antibody disclosed) herein may becompared to release after exposure to a control, e.g., an IgG isotypenegative control, or any other suitable control depending on the TGFβ3inhibitor being tested. Cytokine activation may be assessed inplate-bound (e g , immobilized) and/or soluble assay formats. Levels ofIFNγ, IL-2, IL-1β, TNFα, IL-6, and CCL2 (MCP-1) should not exceed10-fold, e.g., 8-, 6-, 4-, or 2-fold the activation in the negativecontrol. In some embodiments, a positive control may also be used toconfirm cytokine activation in the sample, e.g., in the PBMCs. In someembodiments, these in vitro cytokine release results may be furtherconfirmed in vivo, e.g., in an animal model such as a monkey toxicologystudy, e.g., a 4-week GLP or non-GLP repeat-dose monkey study.

In some embodiments, selection of an antibody or an antigen-bindingfragment thereof for therapeutic use may include: identifying anantibody or antigen-binding fragment that meets the criteria of one ormore of those described herein; carrying out an in vivo efficacy studyin a suitable preclinical model to determine an effective amount of theantibody or the fragment; carrying out an in vivo safety/toxicologystudy in a suitable model to determine an amount of the antibody that issafe or toxic (e.g., MTD, NOAEL, or any art-recognized parameters forevaluating safety/toxicity); and, selecting the antibody or the fragmentthat provides at least a three-fold therapeutic window (preferably6-fold, more preferably a 10-fold therapeutic window, even morepreferably a 15-fold therapeutic window). In certain embodiments, the invivo efficacy study is carried out in two or more suitable preclinicalmodels that recapitulate human conditions. In some embodiments, suchpreclinical models comprise TGFβ1-positive fibrosis. In someembodiments, the preclinical models are selected from liver fibrosismodel, kidney fibrosis model, lung fibrosis model, heart (cardiac)fibrosis model, skin fibrosis model.

Identification of an antibody or antigen-binding fragment thereof fortherapeutic use may further include carrying out an immune safety assay,which may include, but is not limited to, measuring cytokine releaseand/or determining the impact of the antibody or antigen-bindingfragment on platelet binding, activation, and/or aggregation. In certainembodiments, cytokine release may be measured in vitro using PBMCs or invivo using a preclinical model such as non-human primates. In certainembodiments, the antibody or antigen-binding fragment thereof does notinduce a greater than 10-fold release in IL-6, IFNy, and/or TNFa levelsas compared to levels in an IgG control sample in the immune safetyassessment. In certain embodiments, assessment of platelet binding,activation, and aggregation may be carried out in vitro using PBMCs. Insome embodiments, the antibody or antigen-binding fragment thereof doesnot induce a more than 10% increase in platelet binding, activation,and/or aggregation as compared to buffer or isotype control in theimmune safety assessment.

The selected antibody or the fragment may be used in the manufacture ofa pharmaceutical composition comprising the antibody or the fragment.Such pharmaceutical composition may be used in the treatment of a TGFβindication in a subject as described herein. For example, the TGFβindication may be a fibrotic disorder, such as organ fibrosis, e.g.,liver fibrosis. Thus, the invention includes a method for manufacturinga pharmaceutical composition comprising a TGFβ inhibitor, wherein themethod includes the step of selecting a TGFβ inhibitor which is testedfor immune safety as assessed by immune safety assessment comprising acytokine release assay and optionally further comprising a plateletassay. The TGFβ inhibitor selected by the method does not triggerunacceptable levels of cytokine release, as compared to control (such asIgG control). Similarly, the TGFβ inhibitor selected by the method doesnot cause unacceptable levels of platelet aggregation, plateletactivation and/or platelet binding. Such TGFβ inhibitor is thenmanufactured at large-scale, for example 250L or greater, e.g., 1000L,2000L, 3000L, 4000L or greater, for commercial production of thepharmaceutical composition comprising the TGFβ inhibitor.

Pharmaceutical Compositions

The invention further provides pharmaceutical compositions used as amedicament suitable for administration in human and non-human subjects.One or more antibodies that selectively binds an LTBP1-TGFβ1 complexand/or an LTBP3-TGFβ1 complex can be formulated or admixed with apharmaceutically acceptable carrier (excipient), including, for example,a buffer, to form a pharmaceutical composition. Such formulations may beused for the treatment of a disease or disorder that involves TGFβsignaling or dysregulation thereof. In some embodiments, such disease ordisorder associated with TGFβ signaling involves one or more contexts,i.e., the TGFβ is associated with a particular type or types ofpresenting molecules. In some embodiments, such context occurs in a celltype-specific and/or tissue-specific manner In some embodiments, forexample, such context-dependent action of TGFβ signaling is mediated inpart via GARP, LRRC33, LTBP1 and/or LTBP3.

In some embodiments, the antibody of the present invention bindsselectively to a single context of TGFβ, such that the antibody bindsTGFβ in a complex with LTBP presenting molecules, e.g., LTBP1 and/orLTBP3. Thus, such pharmaceutical compositions may be administered topatients for alleviating a TGFβ-related indication (e.g., fibrosis)associated with TGFβ1 activation/release from LTBP1 and/or LTBP3.

A pharmaceutically “acceptable” carrier (excipient) means that thecarrier is compatible with the active ingredient of the composition (andpreferably, capable of stabilizing the active ingredient) and notdeleterious to the subject to be treated. Examples of pharmaceuticallyacceptable excipients (carriers), including buffers, would be apparentto the skilled artisan and have been described previously. See, e.g.,Remington: The Science and Practice of Pharmacy 20th Ed. (2000)Lippincott Williams and Wilkins, Ed. K. E. Hoover. In one example, apharmaceutical composition described herein contains more than oneantibody that selectively binds a LTBP1-TGFβ1 complex and/or aLTBP3-TGFβ1 complex, where the antibodies recognize differentepitopes/residues of the LTBP1-TGFβ1 complex and/or LTBP3-TGFβ1 complex.

The pharmaceutical compositions to be used in the present methods cancomprise pharmaceutically acceptable carriers, excipients, orstabilizers in the form of lyophilized formulations or aqueous solutions(Remington: The Science and Practice of Pharmacy 20th Ed. (2000)Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations used, and may comprise buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acidand methionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Pharmaceutically acceptable excipients arefurther described herein.

The invention also includes pharmaceutical compositions that comprise anantibody or fragment thereof according to the present invention, and apharmaceutically acceptable excipient.

Thus, the antibody or a molecule comprising an antigen-binding fragmentof such antibody can be formulated into a pharmaceutical compositionsuitable for human administration.

The pharmaceutical formulation may include one or more excipients. Insome embodiments, excipient(s) may be selected from the list provided inthe following:https://www.accessdata.fda.gov/scripts/cderhig/index.Cfm?event=browseByLetter.page&Letter=A

The pharmaceutical composition is typically formulated to a finalconcentration of the active biologic (e,g., monoclonal antibody,engineered binding molecule comprising an antigen-binding fragment,etc.) to be between about 2 mg/mL and about 200 mg/mL. For example, thefinal concentration (wt/vol) of the formulations may range between about2-200, 2-180, 2-160, 2-150, 2-120, 2-100, 2-80, 2-70, 2-60, 2-50, 2-40,5-200, 5-180, 5-160, 5-150, 5-120, 5-100, 5-80, 5-70, 5-60, 5-50, 5-40,10-200, 10-180, 10-160, 10-150, 10-120, 10-100, 10-80, 10-70, 10-60,10-50, 10-40, 20-200, 20-180, 20-160, 20-150, 20-120, 20-100, 20-80,20-70, 20-60, 20-50, 20-40, 30-200, 30-180, 30-160, 30-150, 30-120,30-100, 30-80, 30-70, 30-60, 30-50, 30-40, 40-200, 40-180, 40-160,40-150, 40-120, 40-100, 40-80, 40-70, 40-60, 40-50, 50-200, 50-180,50-160, 50-150, 50-120, 50-100, 50-80, 50-70, 50-60, 60-200, 60-180,60-160, 60-150, 60-120, 60-100, 60-80, 60-70, 70-200, 70-180, 70-160,70-150, 70-120, 70-100, 70-80 mg/mL. In some embodiments, the finalconcentration of the biologic in the formulation is about 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, or 200 mg/mL.

According to some embodiments, the TGFβ inhibitor is administered in anamount of about 3000 mg, 2400 mg, 1600 mg, 800 mg, 240 mg, 80 mg, orless.

The pharmaceutical compositions of the present invention are preferablyformulated with suitable buffers. Suitable buffers include but are notlimited to: phosphate buffer, citric buffer, and histidine buffer.

The final pH of the formulation is typically between pH 5.0 and 8.0. Forexample, the pH of the pharmaceutical composition may be about 5.0, 5.2,5.5, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.4, 7.5, 7.6, or 7.8.

The pharmaceutical composition of the present disclosure may comprise asurfactant, such as nonionic detergent, approved for the use inpharmaceutical formulations. Such surfactants include, for example,polysorbates, such as Polysorbate 20 (Tween-20), Polysorbate 80(Tween-80) and NP-40.

The pharmaceutical composition of the present disclosure may comprise astabilizer. For liquid-protein preparations, stability can be enhancedby selection of pH-buffering salts, and often amino acids can also beused. It is often interactions at the liquid/air interface orliquid/solid interface (with the packaging) that lead to aggregationfollowing adsorption and unfolding of the protein. Suitable stabilizersinclude but are not limited to: sucrose, maltose, sorbitol, as well ascertain amino acids such as histidine, glycine, methionine and arginine.

The pharmaceutical composition of the present disclosure may contain oneor any combinations of the following excipients: Sodium Phosphate,Arginine, Sucrose, Sodium Chloride, Tromethamine, Mannitol, BenzylAlcohol, Histidine, Sucrose, Polysorbate 80, Sodium Citrate, Glycine,Polysorbate 20, Trehalose, Poloxamer 188, Methionine, Trehalose,rhHyaluronidase, Sodium Succinate, Potassium Phosphate, DisodiumEdetate, Sodium Chloride, Potassium Chloride, Maltose, HistidineAcetate, Sorbitol, Pentetic Acid, Human Serum Albumin, Pentetic Acid.

In some embodiments, the pharmaceutical composition of the presentdisclosure may contain a preservative.

The pharmaceutical composition of the present disclosure is typicallypresented as a liquid or a lyophilized form. Typically, the products canbe presented in vial (e.g., glass vial). Products available in syringes,pens, or autoinjectors may be presented as pre-filled liquids in thesecontainer/closure systems.

In some examples, the pharmaceutical composition described hereincomprises liposomes containing an antibody that selectively binds aLTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, which can be preparedby any suitable method, such as described in Epstein et al., Proc. Natl.Acad. Sci. USA 82:3688 (1985); Hwang et al. Proc. Natl. Acad. Sci. USA77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomeswith enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

In some embodiments, liposomes with targeting properties are selected topreferentially deliver or localize the pharmaceutical composition tocertain tissues or cell types. For example, certain nanoparticle-basedcarriers with bone marrow-targeting properties may be employed, e.g.,lipid-based nanoparticles or liposomes. See, for example, Sou (2012)“Advanced drug carriers targeting bone marrow”, ResearchGate publication232725109.

The antibodies that selectively bind a LTBP1-TGFβ1 complex and/or aLTBP3-TGFβ1 complex may also be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Exemplary techniques have been described previously, see, e.g.,Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing(2000).

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeutic antibodycompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosageforms such as tablets, pills, capsules, powders, granules, solutions orsuspensions, or suppositories, for oral, parenteral or rectaladministration, or administration by inhalation or insufflation.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g., TWEEEN™ 20, 40, 60, 80 or 85)and other sorbitans (e.g., SPAN™ 20, 40, 60, 80 or 85). Compositionswith a surface-active agent will conveniently comprise between 0.05 and5% surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as INTRALIPID™, LIPSYN™, INFONUTROL™, LIPOFUNDIN™ andLIPIPHYSAN™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g., egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%.

The emulsion compositions can be those prepared by mixing an antibodythat selectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1complex with Intralipid™ or the components thereof (soybean oil, eggphospholipids, glycerol and water).

Use of Inhibitors that Selectively Bind a LTBP1/3-TGFβ1 Complex

The inhibitors, e.g., antibodies and antigen-binding portions thereof,described herein that selectively bind a LTBP1/3-TGFβ1 complex can beused in a wide variety of applications in which modulation of TGFβ1activity associated with LTBP1 or LTBP3 is desired.

In one embodiment, the invention provides a method of inhibiting TGFβ1activation by exposing a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1complex to an inhibitor, e.g., antibody, or antigen-binding portionthereof, which selectively binds a LTBP1/3-TGFβ1 complex. The foregoingmethod can be performed in vitro, e.g., to inhibit TGFβ1 activation incultured cells. The foregoing method can also be performed in vivo,e.g., in a subject in need of TGFβ1 inhibition, or in an animal model inwhich the effect of TGFβ1 inhibition is to be assessed.

Any inhibitor, e.g., antibody, or antigen-binding portion thereof,described herein which selectively binds a LTBP1-TGFβ1 complex and/or aLTBP3-TGFβ1 complex, and any pharmaceutical composition comprising suchantibody, is suitable for use in the methods of the invention. Forexample, in one embodiment, the inhibitor, e.g., antibody, orantigen-binding portion thereof, selectively binds to a LTBP1-TGFβ1complex and a LTBP3-TGFβ1 complex, but does not bind to one or moretargets selected from LTBP1 alone, mature TGFβ1 alone, a GARP-TGFβ1complex, a LRRC33-TGFβ1 complex, and combinations thereof. Exemplaryinhibitor, e.g., antibodies, can inhibit the release of mature TGFβ1from a LTBP1-proTGFβ1 complex and/or a LTBP3-proTGFβ1 complex, withoutinhibiting the release of mature TGFβ1 from a GARP-proTGFβ1 complexand/or a LRRC33-proTGFβ1 complex.

The antibody, or antigen-binding portion thereof, can, in someembodiments, bind a LTBP1-proTGFβ1 complex and/or a LTBP3-proTGFβ1complex with a dissociation constant (K_(D)) of about 10⁻⁸M or less. Insome embodiments, the antibody, or antigen-binding portion thereof has aK_(D) value of about 10⁻⁹ M or less. In some embodiments, the antibody,or antigen-binding portion thereof has a K_(D) value of about 10⁻¹⁰ M orless (e.g., about 10⁻¹¹ M or less). In some embodiments, the antibody,or antigen-binding portion thereof has a K_(D) value of <10 nM, <5 nM <1nM) towards a LTBP1-proTGFβ1 complex and/or a LTBP3-proTGFβ1 complex asmeasured in a suitable in vitro binding assay such as BLI (e.g., Octet).In one embodiment, the antibody, or antigen-binding portion thereof,comprises at least one (e.g., one, two, or three) heavy chain CDRs shownin Table 5, and/or at least one (e.g., one, two, three) light chain CDRsshown in Table 5. In an exemplary embodiment, the antibody, orantigen-binding portion thereof, comprises a heavy chain variable regioncomprising SEQ ID NO:7, and/or a light chain variable region comprisingSEQ ID NO:8. Antibodies and antigen-binding portions thereof which bindthe same epitope as the foregoing antibodies, and/or which compete forbinding with the foregoing antibodies to LTBP1/3-proTGFβ1, are alsouseful in the methods described herein. Additional features of theantibodies, or antigen-binding portions thereof, that are suitable forpracticing the methods of the invention are described herein.

In one embodiment, contacting a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1complex with the inhibitor, e.g., antibody, or antigen-binding portionthereof, inhibits the release of mature TGFβ1 from the LTBP1-TGFβ1complex and/or the LTBP3-TGFβ1 complex. In one embodiment, saidcontacting does not inhibit the release of mature TGFβ1 from presentingmolecules other than LTBP1 and LTBP3. For example, exposing a GARP-TGFβ1complex or a LRRC33-TGFβ1 complex to a context-specific inhibitor, e.g.,antibody, that selectively binds LTBP1/3-TGFβ1 but does not bind TGFβ1in the context of GARP or LRRC33 will not inhibit the release of matureTGFβ1 from the GARP-TGFβ1 complex or the LRRC33-TGFβ1 complex.

LTBP1 and LTBP3 are generally deposited in the extracellular matrix.Accordingly, in one embodiment, complexes comprising LTBP1-TGFβ1 and/orLTBP3-TGFβ1 are associated with the extracellular matrix, e.g., bound tothe extracellular matrix. In some embodiments, the LTBP1/3-TGFβ1complexes are bound to extracellular matrix comprising fibrillin, and/ora protein containing an RGD motif.

The invention also provides a method of reducing TGFβ1 activation in asubject, by administering to the subject an inhibitor, e.g., antibody,or antigen-binding portion thereof, which selectively binds aLTBP1/3-TGFβ1 complex, as described herein. Any antibody, orantigen-binding portion thereof, described herein which selectivelybinds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, and anypharmaceutical composition comprising such antibody, is suitable for usein the methods of the invention.

Exemplary LTBP1/3 inhibitors, e.g., antibodies, bind a LTBP1/3-TGFβ1complex, and inhibit TGFβ1 activation in a context-specific manner, byinhibiting release of TGFβ1 presented by LTBP1 and LTBP3, withoutinhibiting release of TGFβ1 presented by GARP and/or LRRC33. Suchantibodies are useful for blocking a particular subset of TGFβ1 activityin vivo. In one embodiment, the context-specific antibodies providedherein can be used to inhibit TGFβ1 localized to the extracellularmatrix. In another embodiment, the context-specific antibodies caninhibit TGFβ1 without modulating TGFβ1-associated immune activity orimmune response, which is primarily mediated by TGFβ1 presented by GARPand LRRC33. In another embodiment, the context-specific antibodies canbe used to inhibit TGFβ1 activity associated with the extracellularmatrix (e.g., LTBP1-associated TGFβ1 activity and LTBP3-associated TGFβ1activity) without modulating TGFβ1 activity associated withhematopoietic cells, e.g., hematopoietic cells that express GARP and/orLRRC33.

Clinical Applications

Applicant previously described so-called “context-independent”inhibitors of TGFβ1 (see, for example: PCT/US2017/021972 andPCT/US2018/012601) which may be useful for treating various diseases anddisorders involving TGFβ1 dysregulation, including, but are not limitedto, cancer and fibrosis. Unlike traditional TGFβ1 antagonists, thesecontext-independent TGFβ1 inhibitors are capable of selectivelytargeting the TGFβ1 isoform. Within the multifaceted biologicalfunctions driven by the TGFβ1 isoform, however, the context-independentinhibitors do not discriminate tissue-specific (thus context-specific)proTGFβ1 complexes, such that such inhibitors are capable of binding andthereby inhibiting release or activation of mature growth factor fromany of the presenting molecule-proTGFβ1 complexes.

Based at least in part on the recognition that it may be advantageous toprovide even greater selectivity in targeting only a subset of TGFβactivities, context-selective inhibitors of the present disclosure havebeen generated. It is contemplated that by further narrowing particularbiological contexts in which to inhibit TGFβ function, greater safetymay be achieved in a subset of disease conditions or patientpopulations. Specifically, the inventors of the present invention haverecognized that in certain conditions, systemic perturbation of immuneregulation may be particularly undesirable. Because TGFβ plays animportant role in mediating immune response and maintaining immunehomeostasis, broad inhibition of TGFβ activities effectuated in acontext-independent manner may lead to unwanted side effects withoutjustifiable benefits. In these circumstances, it is envisaged that it isadvantageous to specifically target and inhibit matrix-associated TGFβfunction using a context-selective inhibitor, such as those encompassedherein, which does not inhibit the immune components of TGFβ1 function.

Accordingly, the context-specific antibodies can be used to inhibitLTBP1/3-associated TGFβ activity in applications in which TGFβactivation in the context of LTBP1 or LTBP3 is desirable, and in whichTGFβ activation in the context of GARP and/or or LRRC33 is detrimental.

The disease may involve dysregulation or impairment of ECM components orfunction and comprises increased collagen deposition. In someembodiments, the dysregulation or impairment of ECM components orfunction may further comprise increased stiffness and/or ECMreorganization. In some embodiments, the dysregulation or impairment ofECM components or function includes increased myofibroblast cells withinthe disease site. In some embodiments, the dysregulation of the ECMincludes increased stiffness of the matrix, which is implicated in thepathogenesis and/or disease progression of a variety of fibroticconditions and tumors. In some embodiments, the dysregulation of the ECMinvolves fibronectin and/or fibrillin.

Rationale for the Development of Matrix-Targeted TGFβ Inhibitors that donot Inhibit GARP-Associated TGFβ

The invention includes context-specific inhibitors of LTBP1-associatedand/or LTBP3-associated TGFβ. Such inhibitors therefore are capable ofspecifically targeting the ECM-associated latent TGFβ complexes (e.g.,LTBP1-proTGFβ1 and/or LTBP3-proTGFβ1) thereby inhibiting the release ofmature TGFβ growth factor from the latent complex at diseaseenvironments, e.g., fibrotic tissues. Such inhibitors show nosignificant binding activities towards a GARP-proTGFβ1 complex, therebyminimizing unwanted systemic immune modulations. Such antibodies may beadvantageous for use in the treatment of conditions with ECMdysregulation, such as abnormal remodeling and/or stiffness of the ECM.

The context-selective antibodies provided herein may be used in thetreatment of a condition where it is undesirable to stimulate thesubject's immune response and/or in situations where the subject isexpected to benefit from a long-term TGFβ inhibition therapy to manage achronic condition, such as many types of fibrosis.

At least three bases for supporting potential benefits of a TGFβinhibitor that does not target the GARP-proTGFβ1 complex expressed onregulatory T cells are discussed below.

First, GARP-expressing T regulatory cells are a component of the immunesystem that suppress or dampen immune responses of other cells. Thisnotion may be referred to as “tolerance.” This is an important“self-check” built into the immune system to prevent excessive reactionsthat in some situations can result in life-threatening conditions, suchas sepsis, cytokine release syndrome and cytokine storm. TGFβ inhibitiontherapies that exert Treg-inhibitory effects may, therefore, posecertain risk when the normal Treg function is impaired, particularly fora prolonged duration of time, e.g., therapeutic regimen involvingtreatment of six months or longer, and chronic treatment that isadministered for an indefinite period of time. For this reason, patientsin need of TGFβ inhibition therapies, particularly to avoid the risk ofeliciting autoimmunity, may benefit from TGFβ1 inhibitors that do notdirectly perturb the normal Treg function. For example, patientpopulations in need of a long-term TGFβ inhibition therapy may includethose with genetic or congenital conditions, such as DMD, CF and others.In addition, patient populations that suffer from conditions thatinclude inflammation may benefit from a context-specific inhibitor thatdoes not perturb the GARP/Treg function so as to minimize the risk ofexacerbating the existing inflammatory conditions.

Second, increasing evidence points to a link between disproportionateTh17/Treg ratios and pathologies involving inflammation and/or fibrosis.It is generally accepted that the differentiation of the two cell types,Th17 and Treg, is negatively regulated with an inverse relationship.TGFβ1 appears to be a master gatekeeper of this process, such that,TGFβ1 exposure promotes naïve T cells to differentiate into Foxp3+Tregs,whereas TGFβ1 in combination with IL-6, promotes naïve T cells todifferentiate into RORyt+Th17 cells instead. In addition, oncedifferentiated, these cell populations negatively regulate each other.

Lines of evidence suggest that an imbalance in Th17/Treg ratioscorrelates with the pathogenesis and/or progression of fibroticconditions involving chronic inflammation, or severity thereof.

For example, Shoukry et al. reported that Th17 cytokines drive liverfibrosis by regulating TGFβ signaling. The authors examined ex vivo thefrequency of Th17 and Treg populations in liver biopsy samples and foundthat increased Th17/Treg ratio correlated with advanced fibrosis, ascompared to moderate fibrosis or healthy tissue samples. Consistent withthe observation, a strong bias towards Th17 cytokines, IL-22 inparticular, was also detected in fibrotic livers. These data suggestthat increased Th17/Treg ratios lead to an imbalance in pro-fibroticTh17 cytokines, which correlate with severity of liver fibrosis.

Similar inverse correlations of Th17 and Treg populations are observedin other diseases.

For example, increased muscle expression of IL-17 has been reported inpatients with Duchenne muscular dystrophy (DMD), which is a conditionthat manifests chronic inflammation. De Pasquale et al. (Neurology78(17): 1309-14) found that DMD muscle biopsy samples contained higherlevels of IL-17 (a Th17 marker) and lower levels of Foxp3 (a Tregmarker) mRNA compared to control. Elevations in other proinflammatorycytokines, such as TNF-a and MCP-1, were also observed and were found tobe associated with worse clinical outcome of patients. The authorsconcluded that the data point to a possible pathogenic role of IL-17.

Similarly, Jamshidian et al. (J Neuroimmunol 2013, 262(1-2): 106-12)reported biased Treg/Th17 balance away from regulatory towardinflammatory phenotype in patients with relapsed multiple sclerosis andits correlation with severity of clinical symptoms.

A role of regulatory T cells is also implicated in the pathogenesis ofcystic fibrosis (CF). In particular, CF lungs affected by the diseaseare associated with exaggerated Th17 and Th2 cell responses, indicativeof a classic inflammatory phenotype, but also with a deficiency innumbers or function (i.e., impairment) of Treg cells (McGuire (2015) AmJ Respir Crit Care Med 191(8): 866-8).

Furthermore, Zhuang et al. (Scientific Reports (2017) 7: 40141) foundimbalance of Th17/Treg cells in patients with acute anteir uveitis(anterior segment intraocular inflammation with the positive of humanclass I major histocompatibility complex), in which both a markedincrease in Th17 cells and a marked decrease in Treg cells were seen.

Taken together, the inventors of the present disclosure recognized thatwhat appears to be a common feature in these various diseases associatedwith elevated Th17/Treg rations is that the patient suffers from afibrotic condition accompanied by an inflammatory component.

Thus, it is envisaged in the present disclosure that TGFβ inhibitiontherapy that spares the Treg/GARP-arm of the TGFβ function may beparticularly advantageous for an effective treatment of diseasescharacterized by an elevated level of Th17/Treg ratios. In this way, thecontext-selective inhibitors of TGFβ according to the invention areaimed to avoid more systemic effects of TGFβ inhibition that mayinterfere with Treg function, which may lead to exacerbation of existingfibrotic/inflammatory conditions in patients. Thus, the matrix-targetedTGFβ inhibitors described herein are used in a method for treating apatient who has or at risk of developing a fibrotic disorder thatcomprises inflammation. In some embodiments, the patient has an elevatedTh17-to-Treg cell ratio. In some embodiments, the elevated Th17/Tregratio may be predominantly caused by an increased number of Th17 cells,while in other embodiments, the elevated Th17/Treg ratio may bepredominantly caused by a decreased number of Treg cells in the patient(or a biological sample collected from the patient). Yet in furtherembodiments, the elevated Th17/Treg ratio may be caused by a combinationof an increased number of Th17 cells and a decreased number of Tregcells. In some embodiments, elevated levels of IL-17 and/or IL-22detected in patients (or measured in samples collected from thepatients) are also indicative of fibrotic conditions accompanied bychronic inflammation. Such patients may be therefore selected ascandidates for receiving a context-selective TGFβ1 inhibitor therapydisclosed herein.

The third line of reasoning for keeping the GARP-TGFβ1 axis intact in aTGFβ inhibition therapy relates to the benefit of maintaining normalTreg function. As mentioned, GARP is expressed on the cell surface ofTregs and are thought to play a role in TGFβ-mediated immunomodulation.Because Tregs are indispensable for immune homeostasis and theprevention of autoimmunity, unnecessary perturbation of which may putcertain patient populations at higher risk of, for example, infections(reviewed, for example, by: Richert-Spuhler and Lund (2015) Prog MolBiol Transl Sci.

The third line of reasoning for keeping the GARP-TGFβ1 axis intact in aTGFβ inhibition therapy is that regulatory T cells function as a “break”to modulate or dampen over-reactive immune response. The discovery ofFoxp3 as the master regulator of Treg cell development and function wascritical for the understanding of Treg cell biology. Inactivatingmutations in Foxp3 result in the spontaneous development of severeautoimmunity with a scurfy phenotype in mice and IPEX syndrome (‘immunedysregulation, polyendocrinopathy, enteropathy, X-linked’) in humans(see Dominguez-Villear and Haler, Nature Immunology 19, 665-673, 2018).Thus, it raises the possibility that TGFb1 therapy that elicitsinhibitory effects of the Treg/GARP arm of TGFb function, especially ina prolonged treatment, may cause or exacerbate autoimmune response.

Increasing evidence suggests that Tregs not only act to dampen overexuberant effector immune responses, they also have the ability topotentiate appropriate immune responses to pathogens, by participatingin pathogen clearance and protection of the host from collateral damage.Such diverse function of Treg cells is particularly apparent in delicatetissues such as the lung, which is constantly exposed to an externalenvironment from which a variety of pathogens and other foreigncomponents (e.g., viral pathogens, bacterial pathogens, fungalpathogens, and allergens) may gain access to host cells.

For example, influenza virus infection elicits a strong proinflammatorycytokine response with abundance immune cell infiltration. In acuteand/or severe infections, such response can cause serious sequelae insusceptible individuals. Tregs provide a mechanism for dampening viralinfection-associated pathology by controlling the magnitude of immuneresponse in the host. Indeed, pathogen-exposed Tregs retain protectiveeffects in adoptive transfer. Moreover, such adoptive transfer of primedTregs have been shown to ameliorate influenza virus-associated morbidityand to prolong survival in severe immunocompromised animal models.

Accordingly, the invention provides use of an ECM-targeted,context-selective TGFβ inhibitor (e.g., LTBP1-selective orLTBP1/3-selective inhibitors of TGFβ1 activation inhibitors) for thetreatment of a disease that involves matrix-associated TGFβdysregulation in a subject. The subject is suffering from or at risk ofan infection. The infection can be viral infections (e.g., influenzavirus, respiratory syncytial virus or RSV, human immunodeficiency virusor HIV, MARS, SARS, herpes simplex virus or HSV, hepatitis A virus orHAV, hepatitis B virus or HBV, hepatitis C virus or HCV, CMV, Denguevirus, lymphocytic choriomeningitis virus, and West Nile virus),bacterial infections (meningitis, Mycobacterium tuberculosis, Listeriamonocytogenes, Citrobacter rodentium, Salmonella, and E. coli), and/orfungal infections (e.g., Candida, Pneumocytis, Aspergillus,Cryptococcus, and Coccidioides).

Typically, high-risk or at-risk populations (individuals that areconsidered particularly susceptible to severe infections orinfection-triggered responses) include pediatric populations (infants,young children, e.g., human individuals under the age of 7); elderlypopulations (those who are 65 years or older); those with compromisedimmune system due to medical condition, health status, life styles suchas smoking, and/or medications with immunosuppressive effects, etc.

For example, certain medications cause weakened immunity, such aschemotherapy, therapies that target hematopoietic cells such as CD33therapy, steroids, immunosuppressants, and statins.

In some embodiments, high-risk or at-risk populations are those withexisting medical conditions, such as those with chronic infections suchas HIV, those with bone marrow transplantation, pre-diabeticindividuals, diabetic individuals, those with autoimmune disorders suchas RA, asthma and allergy.

Thus, matrix-targeted, context-selective TGFβ inhibitors encompassedherein may be particularly advantageous for treating patients whorequire a long-term or chronic TGFβ therapy since in these scenarios itis beneficial to avoid impairment of immune homeostasis and the normalimmune function that provides the ability to respond effectively topossible infections caused by a variety of pathogens such as thoselisted above.

Accordingly, antibodies that selectively bind LTBP-TGFβ (e.g.,LTBP1-TGFβ1 and LTBP3-TGFβ31), and that do not inhibit TGFβ in thecontext of the immune-associated TGFβ presenters GARP and LRRC33, aretherapeutic candidates for the treatment of fibrotic indications such asorgan fibrosis, and are aimed to avoid TGFβ-related global immuneactivation. In one embodiment, the context-specific antibodies can beused to inhibit LTBP1/3-associated TGFβ activity in applications inwhich TGFβ-mediated immune suppression is beneficial, e.g., in a subjectwho has received a transplant, who is a candidate for receiving atransplant, or who is expected to receive a transplant. In someembodiments, the subject has an advanced stage fibrosis and/or a bonemarrow disease. In some embodiments, the subject has or is at risk ofdeveloping an autoimmune disorder.

The foregoing methods can be used to treat a subject having a conditionfor which inhibition or reduction in LTBP-associated TGFβ activity isbeneficial. For example, the subject may have or be at risk fordeveloping a disorder in which extracellular matrix-associated TGFβactivity has been implicated.

Integrin-mediated activation of latent TGFβ in the extracellular matrixis a key contributor to fibrosis. Without wishing to be bound by theory,it is presently understood that integrins, including αVβ6 and αVβ8, cantrigger the release of TGFβ from presenting molecules including LTBP1and LTBP3. Inhibiting release or activation of TGFβ in this context canreduce or eliminate fibrosis, and/or symptoms associated therewith.

As described, LTBP1 and LTBP3 are produced and are depositedextracellularly as components of the ECM, where they can “present” aproTGFβ complex (latent, inactive precursor of TGFβ1 ) within the ECM.Upon stimulation, the LTBP1/3-proTGFβ complex releases the TGFβ growthfactor (the active, mature form of growth factor) which in turn isthought to be involved in the regulation of the local tissuemicroenvironment, such as ECM maintenance/remodeling and the process offibrosis, possibly by responding to various cytokines, chemokines andgrowth factors, and by interacting with other ECM components, such asfibronectin, Fibrillin, collagen, elastin, and matrix metallopeptidases(MMPs).

In the normal wound healing process that occurs in response to aninjury, for example, TGFβ is thought to facilitate granular tissueformation, angiogenesis, and collagen synthesis and production. TGFβsignaling is also implicated in abnormal tissue fibrogenesis (i.e.,fibrosis), which results in formation of excess fibrous connectivetissue in an organ or tissue in a reparative or reactive processcharacterized by the pathological accumulation of extracellular matrix(ECM) components, such as collagens. In these and other situations, theTGFβ axis may affect further aspects (in addition to fibrotic aspect),such as inflammation, recruitment and phenotypic switch of various celltypes, which may be mediated by its interaction with one or more of theother presenting molecules, such as GARP/LRRC32 and LRRC33. In certaininstances, it is advantageous to preferentially inhibit theLTBP1/3-context of TGFβ activation, without significantly inhibiting oneor more of the other contexts of TGFβ1 activation, in situations whereECM-associated TGFβ that drives fibrosis is to be selectively inhibited.

Accordingly, in one embodiment, the invention provides a method ofreducing TGFβ activation in a subject having, or at risk of developing,a fibrotic disorder by administering to the subject an antibody, orantigen-binding portion thereof, which selectively binds a LTBP1/3-TGFβcomplex, as described herein. In another embodiment, the inventionprovides a method of treating a fibrotic disorder by administering tothe subject an antibody, or antigen-binding portion thereof, whichselectively binds a LTBP1/3-TGFβ complex, as described herein.

In one embodiment, the fibrotic disorder is an organ fibrosis, whereinoptionally, the organ fibrosis is an advanced organ fibrosis. In afurther embodiment, the organ fibrosis is selected from the groupconsisting of kidney fibrosis, liver fibrosis, lung fibrosis, cardiacfibrosis, pancreatic fibrosis, skin fibrosis, scleroderma, musclefibrosis, uterine fibrosis and endometriosis. In another furtherembodiment, the fibrotic disorder comprising chronic inflammation is amuscular dystrophy, multiple sclerosis (MS), or Cystic Fibrosis (CF). Ina further embodiment, the muscular dystrophy is Duchenne musculardystrophy (DMD). In another further embodiment, the MS comprisesperivascular fibrosis. In a further embodiment, the lung fibrosis isidiopathic pulmonary fibrosis (IPF). In another further embodiment, thesubject has chronic kidney disease (CKD). In another embodiment, thesubject has nonalcoholic steatohepatitis (NASH).

In exemplary embodiments, the fibrotic disorder is fibrosis, Alportsyndrome, fibroids, desmoplasia, amyotrophic lateral sclerosis (ALS), orDuchenne muscular dystrophy (DMD).

In one embodiment, the subject has desmoplasia.

In one embodiment, the subject has organ fibrosis, for example, kidneyfibrosis (e.g., fibrosis associated with chronic kidney disease (CKD)),liver fibrosis (e.g., fibrosis associated with nonalcoholicsteatohepatitis (NASH)), lung fibrosis (e.g., idiopathic pulmonaryfibrosis (IPF)), cardiac fibrosis, and/or skin fibrosis (e.g.,scleroderma). In some embodiments, the subject can have advanced organfibrosis. For example, the subject may be in need of an organtransplant. In one embodiment, the subject may be in need of an organtransplant, and the compounds and compositions described herein areadministered to prevent allograft fibrosis from developing in thesubject following receipt of the transplant.

A recent study examined whether inhibiting integrin αVβ6 could preventTGFβ-mediated allograft fibrosis after kidney transplantation (Lo etal., Am. J. Transplant. (2013), 13:3085-3093). Surprisingly, animalstreated with an inhibitory anti-αVβ6 antibody experienced a significantdecrease in rejection-free survival compared to placebo animals. Theauthors conclude that this result cautions against TGFβ inhibition inkidney transplantation, because the immunosuppressive properties of TGFβhelp prevent allograft rejection. The inhibitors, e.g., antibodies, andantigen-binding portions thereof, described herein advantageouslyinhibit activation of TGFβ presented by LTBP1 or LTBP3 in theextracellular matrix, but do not inhibit activation of TGFβ presented byGARP or LRRC33 on immune cells. Accordingly, the context-specificLTBP1/3-TGFβ inhibitors, e.g., antibodies, described herein can preventor reduce allograft fibrosis, without eliminating the immunosuppressiveproperties of TGFβ that are useful for preventing allograft rejection.Accordingly, in one aspect, the invention provides a method for treatinga fibrotic disorder in a subject, comprising administering to thesubject a therapeutically effective amount of an inhibitor of TGFβsignaling, wherein the inhibitor is a selective inhibitor ofECM-associated TGFβ; and, wherein the subject benefits from suppressedimmunity. In one embodiment, the subject has a fibrotic condition andwould benefit from an allograft transplant, or has received an allografttransplant.

Additional fibrotic conditions for which antibodies and/or compositionsof the present disclosure may be used therapeutically include, but arenot limited to, lung indications (e.g., idiopathic pulmonary fibrosis(IPF), chronic obstructive pulmonary disorder (COPD), allergic asthma,cystic fibrosis (CF), acute lung injury, eosinophilic esophagitis,pulmonary arterial hypertension and chemical gas-injury), kidneyindications (e.g., diabetic glomerulosclerosis, focal segmentalglomeruloclerosis (FSGS), chronic kidney disease, fibrosis associatedwith kidney transplantation and chronic rejection, IgA nephropathy, andhemolytic uremic syndrome), liver fibrosis (e.g., non-alcoholicsteatohepatitis (NASH), chronic viral hepatitis, parasitemia, inbornerrors of metabolism, toxin-mediated fibrosis, such as alcohol fibrosis,non-alcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC),primary biliary cirrhosis, and sclerosing cholangitis), cardiovascularfibrosis (e.g., cardiomyopathy, hypertrophic cardiomyopathy,atherosclerosis and restenosis,) systemic sclerosis, skin fibrosis(e.g., skin fibrosis in systemic sclerosis, diffuse cutaneous systemicsclerosis, scleroderma, pathological skin scarring, keloid,post-surgical scarring, scar revision surgery, radiation-inducedscarring and chronic wounds), eye-related conditions such as subretinalfibrosis, uveitis syndrome, uveitis associated with idiopathicretroperitoneal fibrosis, extraocular muscle fibrosis, eye diseasesassociated with the major histocompatibility complex (MHC class I) orhistocompatibility antigens, subretinal fibrosis in macular degeneration(e.g., age-related macular degeneration) and cancers or secondaryfibrosis (e.g., myelofibrosis, head and neck cancer, M7 acutemegakaryoblastic leukemia and mucositis). Other diseases, disorders orconditions related to fibrosis that may be treated using compoundsand/or compositions of the present disclosure, include, but are notlimited to Marfan's syndrome, stiff skin syndrome, scleroderma,rheumatoid arthritis, bone marrow fibrosis, Crohn's disease, ulcerativecolitis, systemic lupus erythematosus, muscular dystrophy, (such asDMD), Dupuytren's contracture, Camurati-Engelmann disease, neuralscarring, dementia, proliferative vitreoretinopathy, corneal injury,complications after glaucoma drainage surgery, and multiple sclerosis(MS). Many such fibrotic indications are also associated withinflammation of the affected tissue(s), indicating involvement of animmune component. Such inflammation may be accompanied by aberrantimmune cell populations, such as increased numbers of Th17 cells,reduced numbers of Treg cells, and/or both. In each case, the affectedpatient may exhibit increased Th17/Treg cell ratios. In someembodiments, diseases to be treated with an antibody according to thepresent disclosure include metabolic disorders, such as metabolic liverdisorders. Non-limiting examples of metabolic disorders include NASH,NAFLD, type 2 diabetes and obesity. In some embodiments, the disease tobe treated with an antibody according to the present disclosure isaortic stenosis.

In another aspect, the invention provides a method of selecting anisoform-specific TGFβ1 inhibitor for the treatment of a fibroticdisorder in a subject, comprising: (a) determining whether the subjectmanifests clinical presentations including fibrosis and one or more ofthe following: (i) inflammation; (ii) immune suppression; (iii)proliferative dysregulation; (iv) need for an allograft transplant; (v)at risk of severe infection; (vi) in need of a long-term TGFβ1inhibition therapy; and (vii) manifestation of an autoimmuneconditions(s); and (b) selecting an isoform-specific, context-dependentTGFβ1 inhibitor or an isoform-specific, context-independent TGFβ1inhibitor for treatment of the fibrotic disorder based on the clinicalpresentations determined in step (a).

In another aspect, the invention provides a method of treating a subjecthaving a fibrotic disorder, comprising (a) selecting a treatment regimencomprising an isoform-specific TGFβ1 inhibitor for the subject, saidselection comprising (i) determining whether the fibrotic disordermanifests clinical presentations including fibrosis and one or more ofthe following: inflammation, immune suppression, proliferativedysregulation, and need for an allograft transplant; and (ii) selectinga treatment regimen comprising an isoform-specific, context-dependentTGFβ1 inhibitor or an isoform-specific, context-independent TGFβ1inhibitor, based on the clinical presentations determined in step (i);and (b) administering the selected treatment regimen to the subject.

In one embodiment of the foregoing aspects, the fibrotic disordermanifests clinical presentations comprising fibrosis, inflammation,immune suppression, and proliferative dysregulation. In an exemplaryembodiment, the fibrotic disorder is myelofibrosis, and the selectedisoform-specific TGFβ1 inhibitor is an isoform-specific,context-independent TGFβ1 inhibitor.

In another embodiment, the fibrotic disorder manifests clinicalpresentations comprising fibrosis, inflammation, and need for anallograft transplant. In one embodiment, the fibrotic disorder manifestsclinical presentations comprising fibrosis and inflammation. In anotherembodiment, the fibrotic disorder is a degenerative disease.

In one embodiment, the fibrotic disorder manifests clinicalpresentations comprising immune suppression and proliferativedysregulation. In an exemplary embodiment, the fibrotic disorder isassociated with a solid tumor, and the selected isoform-specific TGFβ1inhibitor is an isoform-specific LTBP1/3-specific inhibitor and/or aGARP-selective inhibitor. In one embodiment, the solid tumor is amalignant tumor. In another embodiment, the tumor is a benign tumor. Inone embodiment, the subject has desmoplasia, for example, pancreaticdesmoplasia. In another embodiment, the subject has fibroids.

In another aspect, the invention provides a method of treating a subjecthaving a fibrotic disorder with an isoform-specific, LTBP1/3-specificTGFβ1 inhibitor, comprising determining whether the fibrotic disordermanifests clinical presentations including fibrosis and the need for anallograft transplant; and administering an effective amount of anisoform-specific, LTBP1/3-specific TGFβ1 inhibitor to the subject if thefibrotic disorder manifests fibrosis and the need for an allografttransplant.

In another aspect, the invention provides a method of treating a subjecthaving a fibrotic disorder with an isoform-specific, context-independentTGFβ1 inhibitor, comprising determining whether the fibrotic disordermanifests clinical presentations including fibrosis, immune suppressionand/or proliferative dysregulation; and administering an effectiveamount of an isoform-specific, context-independent TGFβ1 inhibitor tothe subject if the fibrotic disorder manifests fibrosis in conjunctionwith immune suppression and/or proliferative dysregulation.

The inhibitors, e.g., antibodies, described herein can be administeredto a subject in an amount effective to treat or reduce symptoms offibrosis. The effective amount of such an inhibitor is an amounteffective to achieve both therapeutic efficacy and clinical safety inthe subject. In one embodiment, an effective amount is an amounteffective to reduce TGFβ1 activity in the extracellular matrix. Inanother embodiment, an effective amount is an amount effective to reducefibrosis in a subject. In another embodiment, the effective amount doesnot inhibit TGFβ1-mediated immune suppression. In some embodiments, suchan inhibitor, e.g., antibody, is a context-specific inhibitor that canblock activation of TGFβ1 that is mediated by an LTBP-containing,ECM-associated TGFβ1. In some embodiments, the LTBP is LTBP1 and/orLTBP3. Assays useful for determining the efficacy of the inhibitors,e.g., antibodies, and/or compositions of the present disclosure for thealteration of fibrosis include, but are not limited to, histologicalassays for counting fibroblasts and basic immunohistochemical analysesknown in the art.

Diseases Involving Proteases:

Activation of TGFβ from its latent complex may be triggered by integrinin a force-dependent manner, and/or by proteases. Evidence suggests thatcertain classes of proteases may be involved in the process, includingbut are not limited to Ser/Thr proteases such as thrombin, Kallikreins,chemotrypsin, elastases, plasmin, as well as zinc metalloproteases ofADAM family such as ADAM 10 and ADAM 17, as well as MMP family, such asMMP-2, MMP-9 and MMP-13. MMP-2 degrades the most abundant component ofthe basement membrane, Collagen IV, raising the possibility that it mayplay a role in ECM-associated TGFβ1 regulation. MMP-9 has beenimplicated to play a central role in tumor progression, angiogenesis,stromal remodeling and metastasis. Thus, protease-dependent activationof TGFβ1 in the ECM may be important for treating cancer.

Kallikreins (KLKs) are trypsin- or chymotrypsin-like serine proteasesthat include plasma Kallikreins and tissue Kallikreins. The ECM plays arole in tissue homeostasis acting as a structural and signaling scaffoldand barrier to suppress malignant outgrowth. KLKs may play a role indegrading ECM proteins and other components which may facilitate tumorexpansion and invasion. For example, KLK1 is highly upregulated incertain breast cancers and can activate pro-MMP-2 and pro-MMP-9. KLK2activates latent TGFβ1, rendering prostate cancer adjacent tofibroblasts permissive to cancer growth. KLK3 has been widely studied asa diagnostic marker for prostate cancer (PSA). KLK3 may directlyactivate TGFβ1 by processing plasminogen into plasmin, whichproteolytically cleaves LAP. KLK6 may be a potential marker forAlzheimer's disease.

Known activators of TGFβ1, such as plasmin, TSP-1 and αVβ6 integrin, allinteract directly with LAP. It is postulated that proteolytic cleavageof LAP may destabilize the LAP-TGFβ interaction, thereby releasingactive TGFβ1. It has been suggested that the region containing54-LSKLRL-59 (SEQ ID NO: 388) is important for maintaining TGFβ1latency. Thus, agents (e.g., antibodies) that stabilize the interaction,or block the proteolytic cleavage of LAP may prevent TGFβ activation.

Many of these proteases associated with pathological conditions (e.g.,cancer) function through distinct mechanisms of action. Thus, targetedinhibition of particular proteases, or combinations of proteases, mayprovide therapeutic benefits for the treatment of conditions involvingthe protease-TGFβ axis. Accordingly, it is contemplated that inhibitors(e.g., TGFβ1 antibodies) that selectively inhibit protease-inducedactivation of TGFβ1 may be advantageous in the treatment of suchdiseases (e.g., cancer). Similarly, selective inhibition of TGFβ1activation by one protease over another protease may also be preferred,depending on the condition being treated.

Plasmin is a serine protease produced as a precursor form calledPlasminogen. Upon release, Plasmin enters circulation and therefore isdetected in serum. Elevated levels of Plasmin appear to correlate withcancer progression, possibly through mechanisms involving disruption ofthe extracellular matrix (e.g., basement membrane and stromal barriers)which facilitates tumor cell motility, invasion, and metastasis. Plasminmay also affect adhesion, proliferation, apoptosis, cancer nutrition,oxygen supply, formation of blood vessels, and activation of VEGF(Didiasova et al., Int. J. Mol. Sci, 2014, 15, 21229-21252). Inaddition, Plasmin may promote the migration of macrophages into thetumor microenvironment (Philips et al., Cancer Res. 2011 Nov 1;71(21):6676-83 and Choong et al., Clin. Orthop. Relat. Res. 2003, 415S,S46-S58). Indeed, tumor-associated macrophages (TAMs) are wellcharacterized drivers of tumorigenesis through their ability to promotetumor growth, invasion, metastasis, and angiogenesis.

Plasmin activities have been primarily tied to the disruption of theECM. However, there is mounting evidence that Plasmin also regulatedownstream MMP and TGF beta activation. Specifically, Plasmin has beensuggested to cause activation of TGF beta through proteolytic cleavageof the Latency Associated Peptide (LAP), which is derived from theN-terminal region of the TGF beta gene product (Horiguchi et al., JBiochem. 2012 Oct; 152(4):321-9), resulting in the release of activegrowth factor. Since TGFβ1 may promote cancer progression, this raisesthe possibility that plasmin-induced activation of TGFb may at least inpart mediate this process.

TGFβ1 has also been shown to regulate expression of uPA, which is acritical player in the conversion of Plasminogen into Plasmin(Santibanez, Juan F., ISRN Dermatology, 2013: 597927). uPA hasindependently been shown to promote cancer progression (e.g., adhesion,proliferation, and migration) by binding to its cell surface receptor(uPAR) and promoting conversion of Plasminogen into Plasmin. Moreover,studies have shown that expression of uPA and/or plasminogen activatorinhibitor-1 (PAI-1) are predictors of poor prognosis in colorectalcancer (D. Q. Seetoo, et al., Journal of Surgical Oncology, vol. 82, no.3, pp. 184-193, 2003), breast cancer (N. Harbeck et al., Clinical BreastCancer, vol. 5, no. 5, pp. 348-352, 2004), and skin cancer (Santibanez,Juan F., ISRN Dermatology, 2013: 597927). Thus, without wishing to bebound by a particular theory, the interplay between Plasmin, TGFβ1, anduPA may create a positive feedback loop towards promoting cancerprogression. Accordingly, inhibitors that selectively inhibitPlasmin-dependent TGFβ1 activation may be particularly suitable for thetreatment of cancers reliant on the Plasmin/TGFβ1 signaling axis.

In one aspect of the invention, the isoform-specific inhibitors of TGFβ1described herein include inhibitors that can inhibit protease-dependentactivation of TGFβ1. In some embodiments, the inhibitors can inhibitprotease-dependent TGFβ1 activation in an integrin-independent manner Insome embodiments, such inhibitors can inhibit TGFβ1 activationirrespective of the mode of activation, e.g., inhibit bothintegrin-dependent activation and protease-dependent activation ofTGFβ1. In some embodiments, the protease is selected from the groupconsisting of: serine proteases, such as Kallikreins, Chemotrypsin,Trypsin, Elastases, Plasmin, as well as zinc metalloproteases (MMPfamily) such as MMP-2, MMP-9 and MMP-13.

In some embodiments, the inhibitors can inhibit Plasmin-inducedactivation of TGFβ1. In some embodiments, the inhibitors can inhibitPlasmin- and integrin-induced TGFβ1 activation. In some embodiments, theinhibitors are monoclonal antibodies that specifically bind TGFβ1. Insome embodiments, the antibody is a monoclonal antibody thatspecifically binds proTGFβ1. In some embodiments, the antibody bindslatent proTGFβ1 thereby inhibiting release of mature growth factor fromthe latent complex. In some embodiments, the high-affinity, LTBP-complexspecific inhibitor of TGFβ1 activation suitable for use in the method ofinhibiting Plasmin-dependent activation of TGFβ1. In some embodiments,the LTBP-complex specific inhibitor of TGFβ1 activation is selected fromAb31, Ab34, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab62,Ab63, and Ab64 (optionally Ab42 or Ab63) (i.e., an antibody orantigen-binding fragment having the heavy and light chain variableregions of the corresponding Ab, as provided herein) avariant/derivative or antigen-binding fragment thereof thereof, or anengineered molecule comprising an antigen-binding fragment thereof. Insome preferred embodiments, the LTBP-complex specific inhibitor of TGFβ1activation is Ab42, a variant/derivative or antigen-binding fragmentthereof, or an engineered molecule comprising an antigen-bindingfragment thereof. In preferred embodiments, the LTBP-complex specificinhibitor of TGFβ1 activation is Ab42 or an antigen-binding fragmentthereof.

In some embodiments, the inhibitor (e.g., TGFβ1 antibody) inhibitscancer cell migration. In some embodiments, the inhibitor inhibitsmacrophage migration. In some embodiments, the inhibitor inhibitsaccumulation of TAMs.

In another aspect, provided herein is a method for treating cancer in asubject in need thereof, the method comprising administering to thesubject an effective amount of an TGFβ1 inhibitor (e.g., TGFβ1antibody), wherein the inhibitor inhibits protease-induced activation ofTGFβ1 (e.g., Plasmin), thereby treating cancer in the subject.

In another aspect, provided herein is a method of reducing tumor growthin a subject in need thereof, the method comprising administering to thesubject an effective amount of an TGFβ1 inhibitor (e.g., TGFβ1antibody), wherein the inhibitor inhibits protease-induced activation ofTGFβ1 (e.g., Plasmin), thereby reducing tumor growth in the subject.

Disease Involving ECM Dysregulation

The extracellular matrix is a cell-secreted network that surrounds cellsand is primarily composed of proteoglycans and fibrous proteins, themost abundant of which is collagen. The novel antibodies disclosedherein may be used in the treatment of diseases associated withextracellular matrix dysregulation. The diseases associated withextracellular matrix dysregulation are typically myofibroblast-drivenpathologies and include cancer, fibrosis, and cardiovascular disease(reviewed, for example, in: Lampi and Reinhart-King (2018) “Targetingextracellular matrix stiffness to attenuate disease: From molecularmechanisms to clinical trials” Sci Tarnsl Med 10(422): eaao0475).Progression of fibrotic conditions involves increased levels of matrixcomponents deposited into the ECM and/or maintenance/remodeling of theECM. TGFβ1 at least in part contributes to this process. This issupported, for example, by the observation that increased deposition ofECM components such as collagens can alter the mechanophysicalproperties of the ECM (e.g., the stiffness of the matrix/substrate) andthis phenomenon is associated with TGFβ1 signaling. The inhibitors ofTGFβ1, such as those described herein may be used to block this processto counter disease progression involving ECM alterations, such asfibrosis. The LTBP-arm of such inhibitors can directly blockECM-associated pro/latent TGFβ3 complexes which are presented by LTBP1and/or LTBP3, thereby preventing activation/release of the growth factorfrom the complex in the disease niche. In some embodiments, theisoform-specific TGFβ1 inhibitors such as those described herein maynormalize ECM stiffness to treat a disease that involvesintegrin-dependent signaling. In some embodiments, the integrincomprises an all chain, 131 chain, or both.

Thus, the antibody may be administered to a subject diagnosed with adisease with extracellular matrix dysregulation in an amount effectiveto treat the disease. Therapeutically effective amount of the antibodymay be an amount sufficient to reduce expression of one or more markersof myofibroblasts, such as a-SMA. The amount may be an amount sufficientto reduce the stiffness of the extracellular matrix of an affectedtissue (e.g., fibrotic tissues). The amount may be an amount sufficientto reduce TGFβ1 downstream effectors, such as phosphorylation of SMAD2and/or SMAD3. In some embodiments, the isoform-selective activationinhibitor of TGFβ1 is selected from Ab31, Ab34, Ab37, Ab38, Ab39, Ab40,Ab41, Ab42, Ab43, Ab44, Ab45, Ab62, Ab63, and Ab64 (optionally Ab42 orAb63) a variant/derivative or antigen-binding fragment thereof thereof,or an engineered molecule comprising an antigen-binding fragmentthereof. In some preferred embodiments, the isoform-selective activationinhibitor of TGFβ1 is Ab42, a variant/derivative or antigen-bindingfragment thereof, or an engineered molecule comprising anantigen-binding fragment thereof. In preferred embodiments, theTGFβ1-selective inhibitor is Ab42 or an antigen-binding fragmentthereof.

Diseases Involving Epithelial-to-Mesenchymal Transition (EMT):

EMT (epithelial mesenchymal transition) is the process by whichepithelial cells with tight junctions switch to mesenchymal properties(phenotypes) such as loose cell-cell contacts. The process is observedin a number of normal biological processes as well as pathologicalsituations, including embryogenesis, wound healing, cancer metastasisand fibrosis (reviewed in, for example, Shiga et al. (2015)“Cancer-Associated Fibroblasts: Their Characteristics and Their Roles inTumor Growth.” Cancers, 7: 2443-2458). Generally, it is believed thatEMT signals are induced mainly by TGFβ. Many types of cancer, forexample, appear to involve transdifferentiation of cells towardsmesenchymal phenotype (such as CAFs) which correlate with poorerprognosis. Thus, LTBP-specific inhibitors of TGFβ1, such as thosedescribed herein, may be used to treat a disease that is initiated ordriven by EMT. Indeed, data exemplified herein (e.g., FIGS. 12 and 13)show that such inhibitors have the ability to suppress expression of CAFmarkers in vivo, such as a-SMA, Coll (Type I collagen), and FN(fibronectin).

Diseases involving Matrix Stiffening and Remodeling

Progression of fibrotic conditions involves increased levels of matrixcomponents deposited into the ECM and/or maintenance/remodeling of theECM. TGFβ1 at least in part contributes to this process. This issupported, for example, by the observation that increased deposition ofECM components such as collagens can alter the mechanophysicalproperties of the ECM (e.g., the stiffness of the matrix/substrate) andthis phenomenon is associated with TGFβ1 signaling. To confirm thisnotion, the present inventors have evaluated the role of matrixstiffness in affecting integrin-dependent activation of TGFβ in primaryfibroblasts transfected with proTGFβ and LTBP1, and grown onsilicon-based substrates with defined stiffness (e.g., 5 kPa, 15 kPa or100 kPa). Matrices with greater stiffness enhance TGFβ1 activation, andthis can be suppressed by antibodies, and antigen-binding portionsthereof, which are capable of binding and thereby inhibiting TGFβ1activation associated with LTBP1/3. These observations suggest thatTGFβ1 influences ECM properties (such as stiffness), which in turn canfurther induce TGFβ1 activation, reflective of disease progression.Thus, antibodies, and antigen-binding portions thereof, that selectivelybind complexes of LTBP1-TGFβ1 and/or LTBP3-TGFβ1, such as thosedescribed herein may be used to block this process to counter diseaseprogression involving ECM alterations, such as fibrosis, tumor growth,invasion, metastasis and desmoplasia. Such inhibitors can directly blockECM-associated pro/latent TGFβ complexes which are presented by LTBP1and/or LTBP3, thereby preventing activation/release of the growth factorfrom the complex in the disease niche.

Fibrosis:

In response to tissue injury or chronic insult due to physicaldamage/trauma, toxic substances, and/or infection, a natural reparativeprocess begins which involves several cell types including fibroblasts,several different types of immune cells, and resident epithelial andendothelial cells. However, if left unchecked, this process can lead toexcessive accumulation of extracellular matrix (ECM) and fibrosis, whichin turn can lead to progressive loss of tissue function and organfailure (Caja et al., Int. J. Mol. Sci. 2018, 19, 1294).

Fibrosis can occur in several different organs, including lung, kidney,liver, heart, and skin. Independent of the organ, the fibrotic responseis characterized by inflammation, altered epithelial-mesenchymalinteractions, and proliferation of fibroblasts. One of the hallmarks offibrosis is the differentiation of fibroblasts into myofibroblasts,which greatly contribute to the dysregulation of the ECM. However,myofibroblasts have also been proposed to come from other cellularsources (e.g., endothelial cells, epithelial cells, and mesenchymal stemcells (Kim, K K et al, Cold Spring Harb. Perspect. Biol., 2017; Okabe,H. Histol. Histophathol., 2016, 31, 141-148; and Li, C et al, NatCommun., 2016, 7, 11455). Moreover, immune cells play an important rolein the process by secreting cytokines and chemokines which promotedifferentiation of myofibroblasts, stimulate ECM deposition, and recruitadditional immune cells to the damaged tissue (Caj a et al., Int. J.Mol. Sci. 2018, 19, 1294).

Similar to fibrotic tissue, activation of cancer-associated fibroblastscan occur in the tumor milieu, which produces excessive amounts of ECM.The ECM provides a scaffold for the infiltration of other cells (e.g.,pro-tumorigenic immune cells) and a substrate for cell migration. Inother cases, excessive ECM may act as a barrier against anti-tumorigenicimmune cells.

TGFβ is recognized as the central orchestrator of the fibrotic response.TGFβ can promote myofibroblast differentiation, recruit immune cells,and affect epithelial and endothelial cell differentiation.Particularly, TGFβ upregulates the production of ECM and basementmembrane proteins, such as fibronectin, collagen, laminin, osteopontin,tenascin, elastin, decorin. TGFβ-induced myofibroblast differentiationcan lead to additional deposition of ECM proteins, secretion of matricmetalloproteinase (MMPs), and myofibroblast proliferation (Fabregat etal, FEBS J. 2016, 283, 2219-2232; Meng et al, Nat. Rev. Nephrol. 2016,12, 325-338; and Kulkarni et al., Am. J. Respir. Cell Mol. Biol., 2016,54, 751-760). Additionally, TGFβ mediates phenotypic changes affectingcontractile proteins and collagen I in vascular smooth muscle cells(VSCM), and can activate myofibroblasts and other stromal cells toenhance the synthesis of collagen cross-linking proteins, such as lysyloxidase (LOX) family of matrix-remodeling enzymes (Busnadiego et al.,Mol. Cell. Biol. 2013, 33, 2388-2401). Moreover, TGFβ has been shown toregulate both EMT and EndMT, which contributes to the differentiation ofpro-fibrotic cell types, such as myofibroblasts and CAFs. Moreover, TGFβhas been shown to induce epithelial apoptosis, which can promote lungand liver fibrosis among other tissues (Barbas-Filho et al., J. Clin.Pathol. 2001, 54, 132-138; and Wang et al., Dev. Dyn. 2017, 247,492-508).

Whether innate or recruited, macrophages are thought to play animportant role in responding to tissue damage and repair. However, uponcertain signals they can become pro-fibrotic. TGFβ, among othercytokines, has also been shown to activate M2 macrophages, which arepro-inflammatory. Upon activation, these macrophages secrete their owncytokines, including TGFβ, ECM components, angiogenic factors, andchemotactic factors. M2 macrophages have been shown to be essential forTGFβ-driven lung fibrosis (Murray et al., Int. J. Biochem. Cell Biol.2011, 43, 154-162).

In light of increasing evidence pointing to the importantce of M2-typemacrophages for disesase progression in many types of fibrosis, aquestion remained as to whether context-selective inhibition ofLTBP1/3-associated TGFβ1 alone (that is, without addressing themacrophage-associated, LRRC33-arm of TGFβ1 activity) might be sufficientto produce a potent anti-fibrotic effect in vivo. Surprisingly, however,data presented herein suggest that selectively targeting thematrix-associated TGFβ1 (e.g., LTBP1/3-proTGFβ31) appears to be just aseffective—if not better—in achieving anti-fibrotic effects in multiplepreclinical models, as targeting all four known LLCs (e.g.,LTBP1/3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1 ) with the use of aso-called context-independent inhibitor of TGFβ1 (see, for example,FIGS. 19 and 20) and do so without triggering T cell stimulationmediated via GARP-proTGFβ1 inhibition. Moreover, previously disclosedLTBP1/3 complex-selective antibodies lacked robust speciescross-reactivity that would be advantageous for both preclinical (e.g.,rodent) and clinical (e.g., human) use. It was not clear whether therare epitopes being sought which would confer both isoform-selectivityand context-selectivity would also enable favorable speciescross-reactivity profiles. Advantageously, novel antibodies disclosedherein possess all of these criteria.

According to the invention, isoform-specific TGFβ1 such as thosedescribed herein are used in the treatment of fibrosis (e.g., fibroticindications, fibrotic conditions) in a subject. Suitable inhibitors tocarry out the present invention include antibodies and/or compositionsaccording to the present disclosure which may be useful for altering orameliorating fibrosis. More specifically, such antibodies and/orcompositions are selective antagonists of TGFβ1 that are capable oftargeting TGFβ1 presented by various types of presenting molecules.TGFβ1 is recognized as the central orchestrator of the fibroticresponse. Antibodies targeting TGFβ decrease fibrosis in numerouspreclinical models. Such antibodies and/or antibody-based compoundsinclude LY2382770 (Eli Lilly, Indianapolis, IN). Also included are thosedescribed in U.S. Pat. Nos. 6,492,497, 7,151,169, 7,723,486 and U.S.Appl. Publ. No. 2011/0008364, the contents of each of which are hereinincorporated by reference in their entirety. Prior art TGFβ antagonistsinclude, for example, agents that target and block integrin-dependentactivation of TGFβ.

However, evidence suggests that such prior art agents may not mediateisoform-specific inhibition and may cause unwanted effects byinadvertently blocking normal function of TGFβ2 and/or TGFβ3. Indeed,data presented herein support this notion. Normal (undiseased) lungtissues contain relatively low but measurable levels of TGFβ2 and TGFβ3,but notably less TGFβ1. In comparison, in certain disease conditionssuch as fibrosis, TGFβ1 becomes preferentially upregulated relative tothe other isoforms. Preferably, TGFβ antagonists for use in thetreatment of such conditions exert their inhibitory activities onlytowards the disease-induced or disease-associated isoform, whilepreserving the function of the other isoforms that are normallyexpressed to mediate tonic signaling in the tissue. Prior art inhibitors(LY2109761, a small molecule TGFβ receptor antagonist, and a monoclonalantibody that targets αVβ6 integrin) both are shown to inhibit TGFβdownstream tonic signaling in non-diseased rat BAL, raising thepossibility that these inhibitors may cause unwanted side effects.Alternatively or additionally, agents that target and blockintegrin-dependent activation of TGFβ may be capable of blocking only asubset of integrins responsible for disease-associated TGFβ1 activation,among numerous integrin types that are expressed by various cell typesand play a role in the pathogenesis. Furthermore, even where suchantagonists may selectively block integrin-mediated activation of theTGFβ1 isoform, it may be ineffective in blocking TGFβ1 activationtriggered by other modes, such as protease-dependent activation.Accordingly, the isoform-specific inhibitors of TGFβ1 such as thosedescribed herein are aimed to prevent the activation step of TGFβ1regardless of the particular mode of activation, while maintainingisoform selectivity.

It is further contemplated that isoform-specific TGFβ1 inhibitors thatpreferentially inhibit matrix-associated over cell-associated antigencomplexes (i.e., display context-bias) may offer a therapeutic advantagein certain clinical situations (e.g., the LTBP-specific inhibitorsdescribed herein). For example, TGFβ1 context-independent inhibitors(which target all four antigen complexes), may increase immuneactivation through the targeting of cell-associated TGFβ1 (e.g.,GARP-TGFβ1 which is expressed on regulatory T cells) Immune activationmay be disadvantageous for certain patients, e.g., patients withautoimmune disease or who are at risk of sepsis. Accordingly,context-bias antibodies may be useful for treating diseases associatewith matrix-associated TGFβ1 complexes (e.g., fibrosis), whileminimizing immune activation.

Previously, it was contemplated that isoform-specific TGFβ3 inhibitorsmight offer an added therapeutic benefit in particular disease states.For example, certain fibrotic diseases to be treated with a TGFβ1inhibitor may also be TGFβ3-positive (i.e., TGFβ31+/TGF133+fibrotictissue) characterized in that the disease tissue (e.g., fibrotic tissue)expresses both the isoforms. Accordingly, the invention includes the useof isoform-selective TGFβ1 inhibitor in conjunction with anisoform-selective TGFβ3 inhibitor in the treatment of such conditions.

Fibrotic indications for which antibodies and/or compositions of thepresent disclosure may be used therapeutically include, but are notlimited to lung indications (e.g., idiopathic pulmonary fibrosis (IPF),chronic obstructive pulmonary disorder (COPD), allergic asthma, acutelung injury, eosinophilic esophagitis, pulmonary arterial hypertensionand chemical gas-injury), kidney indications (e.g., diabeticglomerulosclerosis, focal segmental glomeruloclerosis (FSGS), chronickidney disease (CKD), fibrosis associated with kidney transplantationand chronic rejection, IgA nephropathy, and hemolytic uremic syndrome),liver fibrosis (e.g., non-alcoholic steatohepatitis (NASH), chronicviral hepatitis, parasitemia, inborn errors of metabolism,toxin-mediated fibrosis, such as alcohol fibrosis, non-alcoholicsteatohepatitis-hepatocellular carcinoma (NASH-HCC), primary biliarycirrhosis, and sclerosing cholangitis), cardiovascular fibrosis (e.g.,cardiomyopathy, hypertrophic cardiomyopathy, atherosclerosis andrestenosis,) systemic sclerosis, skin fibrosis (e.g., skin fibrosis insystemic sclerosis, diffuse cutaneous systemic sclerosis, scleroderma,pathological skin scarring, keloid, post-surgical scarring, scarrevision surgery, radiation-induced scarring and chronic wounds),eye-related conditions such as subretinal fibrosis, uveitis syndrome,uveitis associated with idiopathic retroperitoneal fibrosis, extraocularmuscle fibrosis, eye diseases associated with the majorhistocompatibility complex (MHC class I) or histocompatibility antigens,subretinal fibrosis in macular degeneration (e.g., age-related maculardegeneration), and cancers or secondary fibrosis (e.g., myelofibrosis,head and neck cancer, M7 acute megakaryoblastic leukemia and mucositis).Other diseases, disorders or conditions related to fibrosis (includingdegenerative disorders) that may be treated using compounds and/orcompositions of the present disclosure, include, but are not limited toadenomyosis, endometriosis, Marfan's syndrome, stiff skin syndrome,scleroderma, rheumatoid arthritis, bone marrow fibrosis, Crohn'sdisease, ulcerative colitis, systemic lupus erythematosus, musculardystrophy (such as DMD), Parkinson's disease, ALS, Dupuytren'scontracture, Camurati-Engelmann disease, neural scarring, dementia,proliferative vitreoretinopathy, corneal injury, complications afterglaucoma drainage surgery, and multiple sclerosis (MS). Many suchfibrotic indications are also associated with inflammation of theaffected tissue(s), indicating involvement of an immune component. Suchinflammation may be accompanied by aberrant immune cell populations,such as increased numbers of Th17 cells, reduced numbers of Treg cells,and/or both. In each case, the affected patient may exhibit increasedTh17/Treg cell ratios.

In some embodiments, fibrotic indications that may be treated with thecompositions and/or methods described herein include organ fibrosis,such as fibrosis of the lung (e.g., IPF), fibrosis of the kidney (e.g.,fibrosis associated with CKD), fibrosis of the liver, fibrosis of theheart or cardiac tissues, fibrosis of the skin (e.g., scleroderma),fibrosis of the uterus (e.g., endometrium, myometrium), and fibrosis ofthe bone marrow. In some embodiments, such therapy may reduce or delaythe need for organ transplantation in patients. In some embodiments,such therapy may prolong the survival of the patients.

To treat IPF, patients who may benefit from the treatment include thosewith familial IPF and those with sporadic IPF. Administration of atherapeutically effective amount of an isoform-specific inhibitor ofTGFβ1 may reduce myofibroblast accumulation in the lung tissues, reducecollagen deposits, reduce IPF symptoms, improve or maintain lungfunction, and prolong survival. In some embodiments, the inhibitorblocks activation of ECM-associated TGFβ1 (e.g., pro/latent TGFβ1presented by LTBP1/3) within the fibrotic environment of IPF.

Nonalcoholic fatty liver disease (NAFLD) includes a spectrum ofhistological changes that begin with simple fatty infiltration of theliver, also known as simple or isolated steatosis or nonalcoholic fattyliver (NAFL), which may gradually, sometimes over decades, progress tothe development of chronic inflammation (steatohepatitis or NASH),fibrosis, and ultimately cirrhosis. Only a subgroup of patients withNAFL will progress to NASH and subsequent cirrhosis. Currently, thereare no clear criteria to identify this group of patients. NAFLD is themost common cause of chronic liver disease in North America. Currently,there are no approved drugs for the treatment of NASH. Given the highprevalence of NASH, the associated morbidity, the growing burden ofend-stage liver disease, and limited availability of livers for organtransplantation, identifications of therapies that will slow theprogress of, halt, or reverse NASH and NAFLD will address an unmetmedical need.

There is a consensus that TGFβ is a central player in liver fibrosis(reviewed in, for example, Dewidar et al., Cells 2019, 8, 1419, thecontents of which are incorporated herein by reference). Theisoform-specific TGFβ1 inhibitors such as those provided herein (i.e.,isoform-specific inhibitors of TGFβ1 that are selective forLTBP1/3-TGFβ1 complexes, or “matrix-targeted” inhibitors) may be used totreat fibrotic conditions of the liver, such as nonalcoholic fatty liver(NAFL) and fibrosis associated with fatty liver (e.g., NASH). The fattyliver may or may not be inflamed. Inflammation of the liver due to fattyliver (i.e., steatohepatitis) may develop into scarring (fibrosis),which then often progresses to cirrhosis (scarring that distorts thestructure of the liver and impairs its function). The inhibitor maytherefore be used to treat such conditions. In some embodiments, theinhibitor blocks activation of ECM-associated TGFβ1 (e.g., pro/latentTGFβ1 presented by LTBP1/3) within the fibrotic environment of theliver. Administration of the inhibitor in a subject with such conditionsmay reduce one or more symptoms, prevent or retard progression of thedisease, reduce or stabilize fat accumulations in the liver, reducedisease-associated biomarkers (such as serum collagen fragments), reduceliver scarring, reduce liver stiffness, and/or otherwise produceclinically meaningful outcome in a patient population treated with theinhibitor, as compared to a control population not treated with theinhibitor. In some embodiments, an effective amount of the inhibitor mayachieve both reduced liver fat and reduced fibrosis (e.g., scarring) inNASH patients. In some embodiment, an effective amount of the inhibitormay achieve improvement in fibrosis by at least one stage with noworsening steatohepatitis in NASH patients. In some embodiments, aneffective amount of the inhibitor may reduce the rate of occurrence ofliver failure and/or liver cancer in NASH patients. In some embodiments,an effective amount of the inhibitor may normalize, as compared tocontrol, the levels of multiple inflammatory or fibrotic serumbiomarkers as assessed following the start of the therapy, at, forexample, 12-36 weeks. In some embodiments in NASH patients, theisoform-specific TGFβ1 inhibitors may be administered in patients whoreceive one or more additional therapies, including, but are not limitedto myostatin inhibitors, which may generally enhance metabolicregulation in patients with clinical manifestation of metabolicsyndrome, including NASH.

In some embodiments, in NASH or NAFLD patients, the isoform-specific,matrix-targetd, TGFβ1 inhibitors may be administered in patients whoreceive an Acetyl CoA Carboxylase inhibitor (ACCi) (e.g., firsocostat(aka GS-0976) or PF-05221304). Other therapeutics which may be useful incombination with the improved isoform-specific TGFβ1 inhibitorsdescribed herein, include, but are not limited to: GLP-1 receptoragonists or analgues (e.g., semaglutide), farnesoid X receptor (FXR)agonists (e.g., GS-9674; aka Cilofexor), ASK1 inhibitors (e.g.,selonsertib); obeticholic acid, PPAR agonists (e.g., GFT505; akaelafibranor); nitazoxanide, ketohexokinase (KHK) inhibitors (e.g.,PF-06835919); myostatin inhibitors and/or Diacylglycerol0-Acyltransferase 2 (DGAT2) inhibitors (e.g., PF-06865571). In someembodiments, any one or more of the above-mentioned therapeutics can beused in combination with an isoform specific TGFβ1 inhibitor of thepresent disclosure, for example, an isoform-specific TGFβ1 inhibitor incombination with a FXR agonist, an ACC inhibitor, and/or a GLP-1analogue. In some embodiments, TGFβ inhibitors may be used incombination with a myostatin inhibitor in the treatment of a metabolicliver disease in a subject, such as NASH and NAFLD, and liver fibrosisassociated therewith. The subject may also suffer from type 2 diabetesand/or obesity. The TGFβ inhibitors used are preferably TGFβ31-selectiveinhibitors, more preferably context-selective TGFβ1-selective inhibitorsthat target LTBP1/2-associated TGFβ1, such as those disclosed herein.The myostatin inhibitor is preferably a myostatin-selective inhibitor,such as SRK-015 (e.g., see WO2017/218592A1) and trevogrumab, or anyvariant thereof, or an antibody according to WO 2016/098357.

In some embodiments, treatment with the isoform specific TGFβ1inhibitors alone or in combination with one or more additionaltherapeutics reduces hepatic fat as measured by MRI-PDFF. In someembodiments, the reduction of hepatic fat is at least 20%,e.g., >20%, >25%, >30%, >35%, >40%, >45%, or >50%. In some embodiments,treatment with the isoform specific TGFβ1 inhibitors alone or incombination with one or more additional therapeutics reduces serum ALTand/or GGT by at least 20%, e.g., >20%, >25%, >30%, >35%, >40%, >45%,or >50%. In some embodiments, treatment with the isoform specific TGFβ1inhibitors alone or in combination with one or more additionaltherapeutics reduces bile acid synthesis.

In some embodiments, either as monotherapy or in conjunction with one ormore additional therapy (e.g., combination therapy), the TGFβ1inhibitors of the present disclosure may be effective to treat NASH.“Effective treatment” may refer to improvements in hepatic steatosis,liver stiffness, liver biochemistry and serum fibrosis markers. In someembodiments, a 12-week treatment may result in significant decline of atleast 30 percent in hepatic fat measured by magnetic resonanceimaging-proton density fat fraction (MRI-PDFF) from baseline to 12 weeksin at least 50% percent of patients. Improvements in liver biochemistrytests including serum ALT of median relative reduction of at least 25%and GGT of at least 25% along with markers of reduced bile acidsynthesis, may be achieved at 12 weeks.

In some embodiments, the NASH patients may have advanced liver fibrosis(stage F3/F4). In some embodiments, such patients have stage F3 advancedliver fibrosis. In some embodiments, such patients have stage F4 liverfibrosis characterized by cirrhosis. In some embodiments, the NASHpatients develop or at risk of developing hepatocellular carcinomaand/or esophageal varices.

Fibrosis staging in non-alcoholic fatty liver disease according to theclassification derived by the Nonalcoholic Steatohepatitis ClinicalResearch Network Pathology Committee is provided below:

Stages of fibrosis Fibrotic manifestation Fibrosis Stage Perisinusoidalor periportal fibrosis 1 Mild perisinusoidal fibrosis (zone 3) 1AModerate perisinusoidal fibrosis (zone 3) 1B Portal/periportal fibrosis1C Perisinusoidal and portal/periportal fibrosis 2 Bridging fibrosis 3Cirrhosis 4

Therapeutic benefits may by also evaluated by burden of disease andpatient-reported outcomes. NASH also has an impact on quality of lifefor those living with the condition, measured through patient-reportedoutcomes (PROs). PROs may be assessed using tools such as the ChronicLiver Disease Questionnaire (CLDQ-NASH) prior to treatment (e.g.,baseline), particularly those related to physical health-related scores,

Treatment with a TGFβ1 inhibitor (such as LTBP1/3 complex-selectiveinhibitors described herein) either alone (e.g., monotherapy) or incombination with another therapy, may be effective to improve the PROsas compared to the baseline, or as compared to those of populationnorms. In some embodiments, diabetes mellitus may be associated withimpairment in PROs including physical functioning, bodily pain, generalhealth and vitality. Treatment with a TGFβ1 inhibitor (such as LTBP1/3complex-selective inhibitors described herein) either alone (e.g.,monotherapy) or in combination with another therapy, may be effective toimprove physical health-related scores (such as PROs) among subjectswith diabetes (e.g., type 2 diabetes) and/or obesity.

Published studies in the literature suggest that regulatory T cells(Tregs) may play a role in the progression of liver disease intolater-stage fibrosis with greater severity. For example, Zhang et al.reported that persistence of liver cirrhosis is maintained byintrahepatic regulatory T cells that inhibit the process of fibrosisresolution (Transl Res. 2016; 169: 67-79.e1-2). Kobayashi et al.suggested that Tregs are involved in the progression of liver fibrosisinto hepatocellular carcinoma (HCC), a process referred to ashepatocarcinogenesis (Clin Cancer Res. 2007; 13(3): 902-911).

Accordingly, it is contemplated that careful selection of suitable TGFβinhibitor tailored to the disease type and stage of the diseaseprogression should be considered to maximize therapeutic benefit to aparticular patient or patient population.

In some embodiments, a TGFβ1-selective, context-selective inhibitor thattargets matrix-associated TGFβ1 (e.g., LTBP1-proTGFβ1 and/orLTBP3-proTGFβ31), such as those disclosed herein, is selected for use inthe treatment of an early-stage liver disease such as nonalchoholicfatty liver (“NAFL”) and noncirrhotic liver fibrosis associated withNASH. The noncirrhotic liver fibrosis includes liver fibrosis of stages1-3. The TGFβ1-selective, context-selective inhibitor is administered tothe subject in an amount effective to treat the disease, e.g., slow theprogress of, halt, or reverse NAFL and noncirrhotic NASH. Preferably,the effective amount is sufficient to prevent progression to cirrhosisand cirrhosis complications, reduce the need for liver transplantation,and/or improve survival. In some embodiments, efficacy may be shown by,for example, reduction of inflammatory changes, improvement in fibrosis,or both. In some embodiments, the subject has a metabolic condition,such as obesity, type 2 diabetes. The subject with noncirrhotic NASH mayinclude those with a NASH activity score (NAS) greater than or equal to4 with at least 1 point each in inflammation and ballooning along with aNASH Clinical Research Netwoerk (CRN) fibrosis score greater than stage1 fibrosis but less than stage 4 fibrosis. In some embodiments, thetreatment achieves resoluation of steatohepatitis on overallhistopathological reading and no worsening of liver fibrosis on NASH CRNfibrosis score. Resolution of steatohepatitis is defined as absent fattyliver disease or isolated or simple steatosis without steatohepatitisand a NAS score of 0-1 for inflammation, 0 for ballooning, and any valuefor steatosis; or, improvement in liver fibrosis greater than or equalto one stage (NASH CRN fibrosis score) and no worsening ofsteatohepatitis (defined as no increase in NAS for ballooning,inflammation or steatosis); or, both resoluaiton of steatohepatitis andimprovement in fibrosis as defined above.

In some embodiments, a TGFβ1-selective, context-selective inhibitor thattargets matrix-associated TGFβ1 (e.g., LTBP1-proTGFβ1 and/orLTBP3-proTGFβ31), such as those disclosed herein, is selected for use inthe treatment of NASH with compensated cirrhosis. NASH-associatedcompensated cirrhosis is characterized by significant scar formationthat is evident by histopathology, with hepatocytes clustered in nodulessurrounded by dense extracellular matrix. The TGFβ1-selective,context-selective inhibitor is administered to the subject in an amounteffective to halt or slow progression of fibrosis, prevent clinicaldecompensation, reduce the need for liver transplantation, and/orimprove survival.

NASH with decompensated cirrhosis may be characterized by one or more ofthe following criteria: portal hypertension (evidence of portalhypertension may include low platelet counts, esophageal varices,ascites, history of hepatic encephalopathy, splenomegaly); elevatedbilirubin; or elevated international normalized ratio or prolongedprothrombin time. Thus, patients having NASH with compensated cirrhosismay be those not meeting one or more of the aforementioned decompensatedcirrhosis criteria.

In some embodiments, a TGFβ1-selective, context-selective inhibitor thattargets matrix-associated TGFβ1 (e.g., LTBP1-proTGFβ1 and/orLTBP3-proTGFβ31), such as those disclosed herein, is selected for use inthe treatment of NASH with decompensated cirrhosis or HCC associatedwith liver fibrosis. In some embodiments, upon progression of thedisesase into a late-stage or end-stage liver disease characterized bymanifestation of cirrhosis or HCC, the TGFβ1-selective,context-selective inhibitor is replaced with a TGFβ1-selective,context-independent inhibitor capable of targeting bothmatrix-associated and immune cell-associated TGFβ1, e.g.,LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1 (see,for example, WO 2020/014473) in an amount effective to treat livercirrhosis or HCC. In some embodiments, the TGFβ1-selective,context-selective inhibitor and/or the TGFβ1-selective,context-independent inhibitor may be used as monotherapy or inconjunction with one or more additional therapy.

In some embodiments, an effective amount of the inhibitor may normalize,as compared to control, the levels of multiple inflammatory or fibroticserum biomarkers as assessed following the start of the therapy, at, forexample, 12-36 weeks. In some embodiments, inflammatory or fibroticbiomarkers may be used to assess severity of NAFLD (by measure levels ofhepatic steatosis), select patients for treatment, and/or monitordisease progression or treatment response. For example, blood biomarkersand panels may include, but are not limited to:

-   -   i) the Fatty liver index (BMI, waist circumference, serum        triglycerides, and gamma-glutamyltransferase (GGT);    -   ii) the Hepatic steatosis index (serum aspartate        aminotransferase (AST):alanine aminotransferase (ALT) ratio,        BMI, gender, and presence of diabetes mellitus);    -   i) the NAFLD liver fat score (serum ALT, HDL cholesterol,        triglicerides, haemoglobin A_(1c) and leukocyte count);    -   ii) the SteatoTest (BioPredictive) (serum levels of total        bilirubin, GGT, a2-macroglobin, haptoglobin, ALT, apolipoprotein        AI, total cholesterol, triglycerides, glucose (adjusted for age        and gender) and BMI); and    -   iii) the NAFLD ridge score (serum levels of ALT, HDL        cholesterol, triglycerides, haemoglobin A_(1c), leukocyte count,        and comorbidity data (and the presence of hypertension)).

In some embodiments, imaging biomarkers can be used to assess levels ofhepatic steatosis. For example, imaging biomarkers may include but arenot limited to: ultrasonography, controlled attenuation parameter (CAP),MRI-estimated proton density fat fraction (MRI-PDFF), and magneticresonance spectroscopy (MRS).

Liver biopsies are the current standard for diagnosis NASH, however,variability among pathologists limits the effectiveness of suchdiagnostic method. Accordingly, use of the Fatty Liver Inhibition ofProgression (FLIP) algorithm (comprising histological steatosis,activity and fibrosis scores) may be used to improve the consistency ofNASH diagnosis by biopsy. Moreover, many noninvasive biomarkers may alsobe useful for diagnosing and monitoring disease. Accordingly, in someembodiments, inflammatory or fibrotic biomarkers may be used to assessseverity of NASH, select patients for treatment, and/or monitor diseaseprogression or treatment response. Blood biomarkers may include:

-   -   i) apoptosis markers, such as CK18 fragments, total cytokeratin        and sFAS;    -   ii) inflammatory markers, such as CRP, TNF, IL-8, and CXCL10;    -   iii) lipid oxidation products, such as 11-HETE, 9-HODE, 13-HODE,        12-oxo-ODE, LA-13-HODE (oxNASHscore), and 11,12-diHETrE;    -   iv) lysosomal enzymes, such as cathepsin D; and    -   v) combination panels, such as NASHTest (BioPredictive) and NASH        Diagnostics Panel (comprising, presence of diabetes mellitus,        sex, BMI, and serum levels of triglyceride, CK18 fragments, and        total CK18).

In some embodiments, biomarkers and related panels may be useful indiagnosis levels of fibrosis and/or cirrhosis, select patients fortreatment, and/or monitor disease progression or treatment response. Forexample, noninvasive tests of liver fibrosis and cirrhosis include, butare not limited to: AST:ALT ratio, AST:platelet ratio index, fibrosis-4index (age, AST, ALT, and platelet count), NAFLD fibrosis score (age,BMI, impaired fasting glucose and/or diabetes, AST ALT, platelet count,and albumin), BARD score (AST, ALT, BMI, and diabetes).

Specific fibrosis markers and panels may also be useful, and include,but are not limited to: hyaluronic acid; PIIPNP; Pro-C3; TIMP1; Laminin;enhanced liver fibrosis (ELF) panel (PIINP, hyaluronic acid, TIMP1);FibroTest (GGT, total bilirubin, atm, apolipoprotein AI, andhaptoglobin); and FibroMeter NAFLD (body weight, prothrombin index, ALT,AST, ferritin, and fasting glucose).

Imaging biomarkers for liver fibrosis may include, but are not limitedto: FibroScan (TE), point shear wave elastography (pSWE) (aka acousticradiation force impulse (ARFI)), 2D-3D SWE, magnetic resonanceelastography (MRE), and multiparameteric MRI.

Any RGFb-related disease with an inflammatory or auto inflammatoryelement may benefit from the novel inhibitors of the present disclosurewhich spare the regulatory T cell function. Particularly in liverdiseases, e.g., metabolic liver conditions, it may be advantageous toselect a TGFb inhibitor that selectivity target the matrix-associatedTGFb1 signaling.

In one embodiment, the methods and compositions for use as describedherein are useful for treating a subject having primary biliarycholangitis (PBC). In one embodiment, the subject having PBC has beennonresponsive to UDCA (ursodeoxycholic acid) treatment. In oneembodiment, the subject has Barcelona, Paris-I, Toronto, Rotterdam, orParis-II insufficient response to UCDA. In one embodiment, the subjecthaving PBC has ALP≥2xULN (upper limit of normal), and bilirubin >1xULNdespite an at least 1 year therapy with UDCA at the standard recommendeddose (10-15 mg/kg b.w./day).

In another embodiment, the methods and compositions for use as describedherein are useful for treating a subject having primary sclerosingcholangitis (PSC). In one embodiment, the subject having PSC has anelevated ALP, an abnormal cholangiography, endoscopic retrogradecholangiopancreatography, or percutaneous transhepatic cholangiography.In one embodiment, the subject having PSC has a model for end-stageliver disease (MELD) score of at least 14.

In another embodiment, the methods and compositions for use as describedherein are useful for treating a subject having NASH. In one embodiment,a subject has fibrosis stage 2 NASH, fibrosis stage 3 NASH, or fibrosisstage 4 NASH. In one embodiment, a subject has a NAS (NAFLD activityscore) of at least 4, or of at least 5. In one embodiment, a subjectwith NASH has a NAS>4 and fibrosis stage 2 or stage 3. In oneembodiment, a subject with NASH has a NAS>5 and fibrosis stage 2 orstage 3. In one embodiment, a subject with NASH has a model forend-stage liver disease (MELD) score of at least 14.

The isoform-specific TGFβ1 inhibitors such as those provided herein maybe used to treat fibrotic conditions of the kidney, e.g., diseasescharacterized by extracellular matrix accumulation (IgA nephropathy,focal and segmental glomerulosclerosis, crescentic glomerulonephritis,lupus nephritis and diabetic nephropathy) in which significantlyincreased expression of TGFβ in glomeruli and the tubulointerstitium hasbeen observed. While glomerular and tubulointerstitial deposition of twomatrix components induced by TGFβ, fibronectin EDA+and PAI-1, wassignificantly elevated in all diseases with matrix accumulation,correlation analysis has revealed a close relationship primarily withthe TGFβ1 isoform. Accordingly, the isoform-specific TGFβ1 inhibitorsare useful as therapeutic for a spectrum of human glomerular disorders,in which TGFβ is associated with pathological accumulation ofextracellular matrix.

In some embodiments, the fibrotic condition of the kidney is associatedwith chronic kidney disease (CKD). CKD is caused primarily by high bloodpressure or diabetes and claims more than one million lives each year.CKD patients require lifetime medical care that ranges from strict dietsand medications to dialysis and transplants. In some embodiments, theTGFβ1 inhibitor therapy described herein may reduce or delay the needfor dialysis and/or transplantation. In some embodiments, such therapymay reduce the need (e.g., dosage, frequency) for other treatments. Insome embodiments, the isoform-specific TGFβ1 inhibitors may beadministered in patients who receive one or more additional therapies,including, but are not limited to myostatin inhibitors, which maygenerally enhance metabolic regulation in patients with CKD.

Fibrotic conditions that may be treated with the TGFβ1 inhibitor of thepresent disclosure include conditions involving fibrosis and/or chronicinflammation. Such conditions may be neuromuscular disorders, includingbut are not limited to Duchenne muscular dystrophy (DMD), and othergenetic disorders such as multiple sclerosis (MS) and cystic fibrosis(CF). Through the inhibition of both the ECM- and immune cell-associatedTGFβ1 arms, the TGFβ1 inhibitor such as those described herein isthought to suppress fibrotic progression and restore M1/M2 macrophagepolarization.

The organ fibrosis which may be treated with the methods provided hereinincludes cardiac (e.g., cardiovascular) fibrosis. In some embodiments,the cardiac fibrosis is associated with heart failure, e.g., chronicheart failure (CHF). In some embodiments, the heart failure may beassociated with myocardial diseases and/or metabolic diseases. In someembodiments, the isoform-specific, TGFβ1 inhibitors may be administeredin patients who receive one or more additional therapies, including, butare not limited to myostatin inhibitors in patients with cardiacdysfunction that involves heart fibrosis and metabolic disorder.

In some embodiments, fibrotic conditions that may be treated with thecompositions and/or methods described herein include desmoplasia.Desmoplasia may occur around a neoplasm, causing dense fibrosis aroundthe tumor (e.g., desmoplastic stroma), or scar tissue within the abdomenafter abdominal surgery. In some embodiments, desmoplasia is associatedwith malignant tumor. Due to its dense formation surrounding themalignancy, conventional anti-cancer therapeutics (e.g., chemotherapy)may not effectively penetrate to reach cancerous cells for clinicaleffects. Isoform-specific, inhibitors of TGFβ1 such as those describedherein may be used to disrupt the desmoplasia, such that the fibroticformation can be loosened to aid effects of anti-cancer therapy. In someembodiments, the isoform-specific inhibitors of TGFβ1 can be used asmonotherapy (more below).

In some embodiments, a patient has a fibrotic solid tumor (e.g.,desmoplasia) and is or has been excluded from a surgical candidate pool,such that the fibrotic solid tumor is considered to be non-resectable ornon-operable (e.g., risk of surgical intervention outweighs potentialbenefit thereof). Such patient may be a candidate for receiving a TGFβ1inhibition therapy of the present disclosure. The TGFβ1 inhibitiontherapy of the present invention administered to such patients mayrender the tumor become resectable or operable so that the patient maybecome a candidate for surgical resection.

To treat patients with fibrotic conditions, TGFβ1 isoform-specificinhibitors are administered to a subject in an amount effective to treatthe fibrosis. The effective amount of such an antibody is an amounteffective to achieve both therapeutic efficacy and clinical safety inthe subject. In some embodiments, the inhibitor is an antibody that canblock activation of an LTBP-mediated TGFβ1 localized (e.g., tethered) inthe ECM. In some embodiments, the LTBP is LTBP1 and/or LTBP3. In someembodiments, a LTBP-specific inhibitor of TGFβ1 can be combined with aninhibitor of LRRC33-proTGFβ.

Assays useful in determining the efficacy of the antibodies and/orcompositions of the present disclosure for the alteration of fibrosisinclude, but are not limited to, histological assays for countingfibroblasts and basic immunohistochemical analyses known in the art.

In some embodiments, circulating LAP fragment(s) may be used as a serummarker of fibrogenesis. See for example, U.S. Pat. No. 8,198,412, thecontents of which are incorporated herein by reference.

There are many animal models that have been developed to study fibrosis.For example, certain high fat diets in mice has been shown to mimic boththe histopathology and pathogenesis of human NAFLD. Moreover, somegenetic models also display features of human metabolic syndrome andNAFLD, such as db/db and ob/ob mouse models. There are also animalmodels for the study of NASH, which mainly consist of variousdiet-induced models, including, but not limited to, methionine andcholine-deficient diet (MCD), high-cholesterol diet (HCD),choline-deficient high fat diet (CDHFD), choline-deficient L-aminoacid-deficient diet, choline-deficient L-amino acid-deficientdiet+carbon tetrachloride, high-fat diet +streptozotocin, high fat +highcholesterol diet (HFHC), high-fructose diet (HFD), and high-fructosehigh fat diet (HFHF). Genetic mouse models for the study of NASHinclude, but are not limited to foz/foz mice, Hepatocyte-specificPTEN-deficient mice, Db/db mice+diethylnitrosamine (DEN), and db/dbmice+MCD. The details of all of these models, including the pluses andminus of each, are outlined in Jennie Ka Ching Lau et al., J Pathol2017; 241: 36-44; the contents of which are incorporated herein byreference.

Other models useful for testing the efficacy of TGFβ inhibitors infibrosis include the carbon tetrachloride (CCL₄)-induced liver fibrosismodel and adenine-induced kidney fibrosis model.

Another model useful for testing the efficacy of isoform-specific TGFβinhibitors in liver fibrosis include the bile duct ligation (BDL) model(see, e.g., Tag et al., J Vis Exp. 2015; (96): 52438). A useful geneticmodel of kidney fibrosis includes Alport model (discussed elsewhereherein).

In any of such preclinical models, efficacy in fibrosis (e.g.,pro-fibrotic or anti-fibrotic effects in vivo) may be assessed by anysuitable methods, such as: percent of picosirius red (PSR)-positive areain tissue sections; contents (quantification) of hydroxyproline intissue; and immunohistochemical detection and quantification of collAstaining on tissue sections. In some embodiments, histopathologicalappraisal may be carried out, in which fibrosis scoring system can beemployed. Pharmachological effects of test articles (e.g., TGFβinhibitors) may be examined by suitable pharmacodynamics (PD) measures,such as measuring downstream signal transduction events. In case of theTGFβ pathway, for example, suitable PD measure includes relativephosphorylation of SMAD2/3.

Muscle Conditions Associated with Fibrosis

Accumulating evidence indicates that TGFβ plays an important role inmuscle homeostasis, repair, and regeneration. Agents, such as monoclonalantibodies described herein, that selectively modulate LTBP-associatedTGFβ signaling may be effective for treating damaged muscle fibers, suchas in chronic/genetic muscular dystrophies, congenital fibrosis ofocular/extraocular muscles, and acute muscle injuries, without thetoxicities associated with more broadly-acting TGFβ inhibitors.

Accordingly, the present invention provides methods for treating damagedmuscle fibers using an agent that preferentially modulates a subset, butnot all, of TGFβ effects in vivo. Such agents can selectively modulateTGFβ1 signaling (“isoform-specific modulation”) in a particular context,i.e., when presented by LTBP1 or LTBP3.

In skeletal muscle, TGFβ plays a variety of roles including inhibitionof proliferation and differentiation, induction of atrophy, anddevelopment of fibrosis. TGFβ reduces satellite cell proliferation andprevents differentiation (via inhibition of MyoD and myogenin) (Allen,R.E. and L. K. J Cell Physiol, 1987. 133(3): p. 567-72; Brennan, T. J.,et al., Proc Natl Acad Sci USA, 1991. 88(9): p. 3822-6; Massague, J., etal., Proc Natl Acad Sci USA, 1986. 83(21): p. 8206-10; Olson, E. N., etal., J Cell Biol, 1986. 103(5): p. 1799-805). The isoform of TGFβ (i.e.,TGFβ31, 2, or 3) is not specified in these early papers, but is presumedto be TGFβ1. TGFβ also contributes to muscle fibrosis; direct injectionof recombinant TGFβ1 results in skeletal muscle fibrosis, and pan-TGFβinhibition decreases fibrosis in acute and chronically injured muscle(Li, Y., et al., Am J Pathol, 2004. 164(3): p. 1007-19; Mendias, C. L.,et al., Muscle Nerve, 2012. 45(1): p. 55-9; Nelson, C. A., et al., Am JPathol, 2011. 178(6): p. 2611-21). TGFβ1 is expressed by myofibers,macrophages, regulatory T cells, fibroblasts, and fibrocytes within theskeletal muscle (Li, Y., et al., Am J Pathol, 2004. 164(3): p. 1007-19;Lemos, D.R., et al., Nat Med, 2015. 21(7): p. 786-94; Villalta, S. A.,et al., Sci Transl Med, 2014. 6(258): p. 258ra142; Wang, X., et al., JImmunol, 2016. 197(12): p. 4750-4761); and expression is increased uponinjury and in disease (Li, Y., et al., Am J Pathol, 2004. 164(3): p.1007-19; Nelson, C. A., et al., Am J Pathol, 2011. 178(6): p. 2611-21;Bernasconi, P., et al., J Clin Invest, 1995. 96(2): p. 1137-44;Ishitobi, M., et al., Neuroreport, 2000. 11(18): p. 4033-5). TGFβ2 andTGFβ3 are also upregulated (at the mRNA level) in mdx muscle (a mousemodel of Duchenne muscular dystrophy), although to a lesser extent thanTGFβ1 (Nelson, C. A., et al., Am J Pathol, 2011. 178(6): p. 2611-21;Zhou, L., et al., Neuromuscul Disord, 2006. 16(1): p. 32-8). Pessina, etal., recently used lineage tracing experiments to show that cells ofmultiple origins within dystrophic muscle adopt a fibrogenic fate via aTGFβ-dependent pathway (Pessina, P., et al., Stem Cell Reports, 2015.4(6): p. 1046-60).

TGFβ1 has been implicated in human muscular dystrophies. Duchennemuscular dystrophy (DMD) is a severe, progressive, and ultimately fataldisease caused by the absence of dystrophin (Bushby, K., et al., LancetNeurol, 2010. 9(1): p. 77-93). Lack of dystrophin results in increasedsusceptibility to contraction-induced injury, leading to continualmuscle degeneration (Petrof, B. J., et al., Proc Natl Acad Sci USA,1993. 90(8): p. 3710-4; Dellorusso, C., et al., J Muscle Res Cell Motil,2001. 22(5): p. 467-75; Pratt, S.J., et al., Cell Mol Life Sci, 2015.72(1): p. 153-64). Repeated rounds of repair contribute to chronicinflammation, fibrosis, exhaustion of the satellite cell pool, eventualloss of mobility and death (Bushby, K., et al., Lancet Neurol, 2010.9(1): p. 77-93; McDonald, C. M., et al., Muscle Nerve, 2013. 48(3): p.343-56). Expression of TGFβ1 is significantly increased in patients withDMD and correlates with the extent of fibrosis observed in thesepatients (Bernasconi, P., et al., J Clin Invest, 1995. 96(2): p.1137-44; Chen, Y.W., et al., Neurology, 2005. 65(6): p. 826-34).Excessive ECM deposition has detrimental effects on the contractileproperties of the muscle and can limit access to nutrition as themyofibers are isolated from their blood supply (Klingler, W., et al.,Acta Myol, 2012. 31(3): p. 184-95). Recently, additional data hasfurther implicated TGFβ1 in muscular dystrophies. Variants in LTBP4 havebeen found to modify disease severity in mouse and human. In mouse, avariant of LTBP4 is protective in mice lacking dystrophin ory-sarcoglycan (Coley, W.D., et al., Hum Mol Genet, 2016. 25(1): p.130-45; Heydemann, A., et al., J Clin Invest, 2009. 119(12): p.3703-12). In humans, two groups independently identified a variant ofLTBP4 as protective in DMD, delaying loss of ambulation by several years(Flanigan, K.M., et al., Ann Neurol, 2013. 73(4): p. 481-8; van denBergen, J. C., et al., J Neurol Neurosurg Psychiatry, 2015. 86(10): p.1060-5).

Although the nature of the genetic variants in mouse and human differs,in both species the protective variant results in decreased TGFβsignaling (Heydemann, A., et al., J Clin Invest, 2009. 119(12): p.3703-12); Ceco, E., et al., Sci Transl Med, 2014. 6(259): p. 259ra144).Many of the functions of TGFβ1 in skeletal muscle biology have beeninferred from experiments in which purified active growth factor isinjected into animals or added to cells in culture (Massague, J., etal., Proc Natl Acad Sci USA, 1986. 83(21): p. 8206-10; Li, Y., et al.,Am J Pathol, 2004. 164(3): p. 1007-19; Mendias, C. L., et al., MuscleNerve, 2012. 45(1): p. 55-9). Given the importance of cellular contextfor specific functions of TGFβ1 (see, for example, Hinck et al., ColdSpring Harb. Perspect. Biol, 2016. 8(12)) it is possible that some ofthe effects observed in these experiments do not reflect the endogenousrole(s) of the cytokine in vivo. For example, treatment of human dermalfibroblasts with recombinant TGFβ1, myostatin, or GDF11 results innearly identical changes in gene expression in these cells, although invivo the roles of these proteins are quite different (Tanner, J. W.,Khalil, A., Hill, J., Franti, M., MacDonnell, S. M., GrowthDifferentiation Factor 11 Potentiates Myofibroblast Activation, inFibrosis: From Basic Mechanisms to Targeted therapies. 2016: Keystone,Colo.).

Multiple investigators have used inhibitors of TGFβ to clarify the roleof the growth factor in vivo. Treatment of mdx mice with the pan-TGFβneutralizing antibody 1D11 clearly results in reduced fibrosis (byhistology and hydroxyproline content), reduced muscle damage (reducedserum creatine kinase and greater myofiber density), and improved musclefunction (by plethysmography, force generation of isolated EDL muscles,and increased forelimb grip strength) (Nelson, C. A., et al., Am JPathol, 2011. 178(6): p. 2611-21; Andreetta, F., et al., J Neuroimmunol,2006. 175(1-2): p. 77-86; Gumucio, J. P., et al., J Appl Physiol (1985),2013. 115(4): p. 539-45). In addition, myofiber-specific expression of adominant negative TGFβ type II receptor protects against muscle damageafter cardiotoxin injury and in 6-sarcoglycan−/− mice (Accornero, F., etal., Hum Mol Genet, 2014. 23(25): p. 6903-15). The proteoglycan decorin,which is abundant in skeletal muscle and inhibits TGFβ activity,decreases muscle fibrosis in mdx mice and following laceration injury(Li, Y., et al., Mol Ther, 2007. 15(9): p. 1616-22; Gosselin, L. E., etal., Muscle Nerve, 2004. 30(5): p. 645-53). Other molecules with TGFβinhibitory activity, such as suramin (an anti-neoplastic agent) andlosartan (an angiotensin receptor blocker) have been effective inimproving muscle pathology and reducing fibrosis in mouse models ofinjury, Marfan's syndrome, and muscular dystrophy (Spurney, C. F., etal., J Cardiovasc Pharmacol Ther, 2011. 16(1): p. 87-95; Taniguti, A.P., et al., Muscle Nerve, 2011. 43(1): p. 82-7; Bedair, H. S., et al.,Am J Sports Med, 2008. 36(8): p. 1548-54; Cohn, R. D., et al., Nat Med,2007. 13(2): p. 204-10). While all of the therapeutic agents describedabove do inhibit TGFβ1 or its signaling, none of them is specific forthe TGFβ1 isoform. For example, 1D11 binds to and inhibits the TGFβ1, 2,and 3 isoforms (Dasch, J. R., et al., J Immunol, 1989. 142(5): p.1536-41). Suramin inhibits the ability of multiple growth factors tobind to their receptors, including PDGF, FGF, and EGF, in addition toTGFβ1 (Hosang, M., J Cell Biochem, 1985. 29(3): p. 265-73; Olivier, S.,et al., Eur J Cancer, 1990. 26(8): p. 867-71; Scher, H. I. and W. D.Heston, Cancer Treat Res, 1992. 59: p. 131-51). Decorin also inhibitsmyostatin activity, both by direct binding and through upregulation offollistatin, a myostatin inhibitor (Miura, T., et al., Biochem BiophysRes Commun, 2006. 340(2): p. 675-80; Brandan, E., C. Cabello-Verrugio,and C. Vial, Matrix Biol, 2008. 27(8): p. 700-8; Zhu, J., et al., J BiolChem, 2007. 282(35): p. 25852-63). Losartan affects additional signalingpathways through its effects on the renin-angiotensin-aldosteronesystem, including the IGF-1/AKT/mTOR pathway (Burks, T.N., et al., SciTransl Med, 2011. 3(82): p. 82ra37; Sabharwal, R. and M.W. Chapleau, ExpPhysiol, 2014. 99(4): p. 627-31; McIntyre, M., et al., Pharmacol Ther,1997. 74(2): p. 181-94). Therefore, all of these therapies inhibitadditional molecules which may contribute to their therapeutic effects,as well as toxicities.

Apart from chronic inflammation, the hallmark of DMD is excessive, andprogressive, fibrosis. In advanced disease the fibrosis is so severethat it can actually isolate individual muscle fibers from their bloodsupply. It also alters the contractile properties of the muscle. Inhuman patients, there is a strong correlation between the extent ofTGFβ1 upregulation and fibrosis, and a strong link between the extent offibrosis and negative mobility outcomes. Therefore, in some embodiments,LTBP-proTGFβ1 inhibitors may be administered to dystrophic patients forthe prevention and/or reduction of fibrosis to selectively target theECM-associated TGFβ1 effects in the disease. In some embodiments,various isoform- and/or context-selective agents described herein can beemployed to achieve inhibition of TGFβ1 signaling to prevent fibrosisand promote myogenesis, but without having unwanted effects on theimmune system (e.g., through GARP or LRRC33).

Administration

To practice the method disclosed herein, an effective amount of thepharmaceutical composition described above can be administered to asubject (e.g., a human) in need of the treatment via a suitable route,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, inhalation or topical routes. When used in thetreatment of fibrosis and/or metabolic conditions, preferably the TGFβinhibitor is administered subcutaneously. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution. Alternatively, inhibitors, e.g., antibodies, orantigen-binding portions thereof, that selectively bind a LTBP1-TGFβcomplex and/or a LTBP3-TGFβ complex can be aerosolized using afluorocarbon formulation and a metered dose inhaler, or inhaled as alyophilized and milled powder.

The subject to be treated by the methods described herein can be amammal, more preferably a human. Mammals include, but are not limitedto, farm animals, sport animals, pets, primates, horses, dogs, cats,mice and rats. A human subject who needs the treatment may be a humanpatient having, at risk for, or suspected of having a TGFβ-relatedindication, such as those noted above. A subject having a TGFβ-relatedindication can be identified by routine medical examination, e.g.,laboratory tests, organ functional tests, CT scans, or ultrasounds. Asubject suspected of having any of such indication might show one ormore symptoms of the indication. A subject at risk for the indicationcan be a subject having one or more of the risk factors for thatindication.

As used herein, the terms “effective amount” and “effective dose” referto any amount or dose of a compound or composition that is sufficient tofulfill its intended purpose(s), i.e., a desired biological or medicinalresponse in a tissue or subject at an acceptable benefit/risk ratio. Forexample, in certain embodiments of the present invention, the intendedpurpose may be to inhibit TGFβ-1 activation in vivo, to achieveclinically meaningful outcome associated with the TGFβ-1 inhibition.Effective amounts vary, as recognized by those skilled in the art,depending on the particular condition being treated, the severity of thecondition, the individual patient parameters including age, physicalcondition, size, gender and weight, the duration of the treatment, thenature of concurrent therapy (if any), the specific route ofadministration and like factors within the knowledge and expertise ofthe health practitioner. These factors are well known to those ofordinary skill in the art and can be addressed with no more than routineexperimentation. It is generally preferred that a maximum dose of theindividual components or combinations thereof be used, that is, thehighest safe dose according to sound medical judgment. It will beunderstood by those of ordinary skill in the art, however, that apatient may insist upon a lower dose or tolerable dose for medicalreasons, psychological reasons or for virtually any other reasons.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of a TGFβ-related indication. Alternatively, sustainedcontinuous release formulations of an antibody that selectively binds aLTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex may be appropriate.Various formulations and devices for achieving sustained release wouldbe apparent to the skilled artisan and are within the scope of thisdisclosure.

In one example, dosages for an inhibitor, e.g., antibody, thatselectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex asdescribed herein may be determined empirically in individuals who havebeen given one or more administration(s) of the inhibitor. Individualsare given incremental dosages of the inhibitor. To assess efficacy, anindicator of the TGFβ-related indication can be followed. For example,methods for measuring for myofiber damage, myofiber repair, inflammationlevels in muscle, and/or fibrosis levels in muscle are well known to oneof ordinary skill in the art.

The present invention encompasses the recognition that agents capable ofmodulating the activation step of TGFβ s in an isoform-specific manner,and a context-specific manner, may provide improved safety profiles whenused as a medicament. Accordingly, the invention includes inhibitors,e.g., antibodies and antigen-binding fragments thereof, that selectivelybind and inhibit activation of TGFβ1, but not TGFβ2 or TGFβ3, therebyconferring specific inhibition of the TGFβ1 signaling in vivo whileminimizing unwanted side effects from affecting TGFβ2 and/or TGFβ3signaling. Likewise, the invention includes inhibitors, e.g., antibodiesand antigen-binding fragments thereof, that selectively inhibitactivation of TGFβ1 presented by LTBP1 and/or LTBP3, but not TGFβ1presented by GARP or LRRC33, thereby conferring specific inhibition ofLTBP1/3-associated TGFβ1 signaling in vivo while minimizing unwantedside effects caused by modulation of GARP-associated TGFβ1 and/orLRRC33-associated TGFβ1.

In some embodiments, the inhibitors, e.g., antibodies, orantigen-binding portions thereof, as described herein, are not toxicwhen administered to a subject. In some embodiments, the inhibitors,e.g., antibodies, or antigen-binding portions thereof, as describedherein, exhibit reduced toxicity when administered to a subject ascompared to an antibody that binds to both TGFβ1 and TGFβ2. In someembodiments, the inhibitors, e.g., antibodies, or antigen-bindingportions thereof, as described herein, exhibit reduced toxicity whenadministered to a subject as compared to an inhibitor that binds to bothTGFβ1 and TGFβ3. In some embodiments, the inhibitors, e.g., antibodies,or antigen-binding portions thereof, as described herein, exhibitreduced toxicity when administered to a subject as compared to aninhibitor that binds to TGFβ1, TGFβ2 and TGFβ3.

Generally for administration of any of the inhibitors, e.g., antibodies,described herein, an initial candidate dosage can be about 0.5-30 mg/kgper dose, e.g., about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 30 mg/kgper dose. Typically, the composition comprising an antibody or afragment thereof encompassed by the present disclosure is administeredto a human patient at the dosage at suitable intervals, such as once ortwice weekly, every 1-8 weeks, etc. In some embodiments, frequency ofadministration may be adjusted to, for example, twice a week, once aweek, every two weeks, every three weeks, every four weeks, every sixweeks, every eight weeks, etc. For the purpose of the presentdisclosure, a typical daily dosage might range from about any of 1 mg/kgto 20 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toalleviate a TGFβ-related indication, or a symptom thereof.

In one embodiment, the antibody, or antigen-binding fragment thereof, isadministered to the subject at a dosage of between 0.1 and 30 mg/kg,between 0.5 and 30 mg/kg, between 1 and 30 mg/kg, between 5 and 30mg/kg, between 10 and 30 mg/kg, between 15 and 30 mg/kg, between 20 and30 mg/kg, between 25 and 30 mg/kg, between 0.1 and 25 mg/kg, between 0.5and 25 mg/kg, between 1 and 25 mg/kg, between 5 and 25 mg/kg, between 10and 25 mg/kg, between 15 and 25 mg/kg, between 20 and 25 mg/kg, between0.1 and 20 mg/kg, between 0.5 and 20 mg/kg, between 1 and 20 mg/kg,between 5.0 and 20 mg/kg, between 10 and 20 mg/kg, between 15 and 20mg/kg, between 0.1 and 15 mg/kg, between 0.5 and 15 mg/kg, between 1 and15 mg/kg, between 5 and 15 mg/kg, between 10 and 15 mg/kg, between 5.0and 20 mg/kg, between 10 and 20 mg/kg, between 15 and 20 mg/kg, between0.1 and 10 mg/kg, between 0.5 and 10 mg/kg, between 1 and 10 mg/kg,between 5 and 10 mg/kg, optionally, wherein the subject is administeredthe antibody, or antigen-binding portion thereof, twice a week, once aweek, once every 2 weeks, once every 3 weeks, once a month, or everyother month.

An exemplary dosing regimen comprises administering an initial dose ofabout 2 mg/kg, followed by one or more maintenance doses. For example,an initial dose may be between about 2 and 30 mg/kg, for instance, oncea week or twice a week. Thereafter, maintenance dose(s) may follow, forexample, between about 0.1 and 20 mg/kg, for instance, once a week,every other week, once a month, etc. However, other dosage regimens maybe useful, depending on the pattern of pharmacokinetic decay that thepractitioner wishes to achieve. Pharmacokinetics experiments have shownthat the serum concentration of an inhibitor, e.g., antibody, disclosedherein (e.g., SR-AB2) remains stable for at least 7 days afteradministration to a preclinical animal model (e.g., a mouse model).Without wishing to be bound by any particular theory, this stabilitypost-administration may be advantageous since the antibody may beadministered less frequently while maintaining a clinically effectiveserum concentration in the subject to whom the antibody is administered(e.g., a human subject). In some embodiments, dosing frequency is onceevery week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks,every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or onceevery month, every 2 months, or every 3 months, or longer. The progressof this therapy is easily monitored by conventional techniques andassays. The dosing regimen (including the antibody used) can vary overtime.

According to some embodiments, serum concentrations of the LTBPcontext-selective TGFβ1 inhibitor that are therapeutically effective totreat a TGFβ1-related indication in accordance with the presentdisclosure may be at least about 10 μg/mL, e.g., between about 10 μg/mLand 1.0 mg/mL. In some embodiments, effective amounts of the antibody asmeasured by serum concentrations are about 20-400 μg/mL. In someembodiments, effective amounts of the antibody as measured by serumconcentrations are about 100-800 μg/mL. In some embodiments, effectiveamounts of the inhibitor as measured by serum concentrations are atleast about 20 μg/mL, e.g., at least about 50 μg/mL, 100 μg/mL, 150μg/mL or 200 μg/mL. In preferred embodiments, in non-human primates,there are no observed toxicities (for example: no cardiotoxicities,hyperplasia and inflammation, dental and gingival findings) associatedwith such inhibitor after maintaining serum concentration levels ofabout 2,000-3,000 μg/mL for at least 4 weeks, e.g., at least 4 weeks,preferably at least 8 weeks, more preferably at least 12 weeks.Therefore, about 10-100 fold therapeutic window may be achieved.

In some embodiments, for an adult patient of normal weight, dosesranging from about 0.3 to 5.00 mg/kg may be administered. The particulardosage regimen, e.g., dose, timing and repetition, will depend on theparticular individual and that individual's medical history, as well asthe properties of the individual agents (such as the half-life of theagent, and other relevant considerations).

For the purpose of the present disclosure, the appropriate dosage of aninhibitor, e.g., antibody or antigen-binding fragment thereof, thatselectively binds a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex willdepend on the specific antibody (or compositions thereof) employed, thetype and severity of the indication, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the inhibitor, and thediscretion of the attending physician. In some embodiments, a clinicianwill administer an inhibitor, e.g., antibody, that selectively binds aLTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, until a dosage isreached that achieves the desired result. Administration of aninhibitor, e.g., antibody, that selectively binds a LTBP1-TGFβ1 complexand/or a LTBP3-TGFβ1 complex can be continuous or intermittent,depending, for example, upon the recipient's physiological condition,whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of an inhibitor, e.g., antibody, that selectively binds aLTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced dose, e.g., either before, during, or after developing aTGFβ-related indication.

Based on the observation that inhibiting TGFβ3 can increase collagendeposition or accumulation in fibrosis, add-on therapy comprising aTGFβ1-selective inhibitor (such as the novel antibodies disclosedherein) may be considered for patients who are treated with a TGFβinhibitor with TGFβ3-inhibiting activity, e.g., inhibitors of TGFβ1/2/3,TGFβ1/3 and TGFβ3. Examples of TGFβ inhibitors with TGFβ3-inhibitingactivity include but are not limited to: low molecular weightantagonists of TGFβ receptors, e.g., ALKS antagonists, such asGalunisertib (LY2157299 monohydrate); monoclonal antibodies (such asneutralizing antibodies) that inhibit all three isoforms(“pan-inhibitor” antibodies) (see, for example, WO 2018/134681);monoclonal antibodies that preferentially inhibit two of the threeisoforms (e.g., TGFβ1/3 (for example WO 2006/116002); and engineeredmolecules (e.g., fusion proteins) such as ligand traps (for example, WO2018/029367; WO 2018/129331 and WO 2018/158727). In some embodiments,the ligand trap comprises the structure in accordance with thedisclosure of WO/2018/15872. In some embodiments, the ligand trapcomprises the structure in accordance with the disclosure of WO2018/029367; WO 2018/129331. In some embodiments, the ligand trap is aconstruct known as M7824. In some embodiments, the ligand trap is aconstruct known as AVID200. In some embodiments, the neutralizingpan-TGFβ antibody is GC1008 or a derivative thereof. In someembodiments, such antibody comprises the sequence in accordance with thedisclosure of WO/2018/134681.

In some embodiments, the antibody is a neutralizing antibody thatspecifically binds both TGFβ1 and TGFβ3. In some embodiments suchantibody preferentially binds TGFβ1 over TGFβ3. For example, theantibody comprises the sequence in accordance with the disclosure ofWO/2006/116002. In some embodiments, the antibody is 21D1.

The add-on therapy is aimed to counter or overcome the pro-fibroticeffect of TGFβ3 inhibition in patients who have received or arereceiving a TGFβ inhibitor with TGFβ3 inhibitory activities. In someembodiments, the patient has a fibrotic disorder or is at risk ofdeveloping a fibrotic disorder. For example, the patient may suffer froma metabolic condition that is associated with higher risk of developingliver fibrosis. The metabolic conditions linked to such risk includeobesity, type 2 diabetes and NASH. Accordingly, the invention includes aTGFβ1-selective inhibitor for use in an add-on therapy of a subjecttreated with a TGFβ3 inhibitor, in an amount sufficient to reducepro-fibrotic effects of the TGFβ3 inhibitor. In some embodiments, thesubject has fibrosis. In some embodiments, the subject hasmyelofibrosis. In some embodiments, the subject has advanced cancer,e.g., metastatic or locally advanced tumor. In some embodiments, theTGFβ1-selective inhibitor is selected from Ab31, Ab34, Ab37, Ab38, Ab39,Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab62, Ab63, and Ab64 (optionallyAb42 or Ab63) a variant/derivative or antigen-binding fragment thereofthereof, or an engineered molecule comprising an antigen-bindingfragment thereof. In some embodiments, the TGFβ1-selective inhibitor isAb42, a variant/derivative or antigen-binding fragment thereof, or anengineered molecule comprising an antigen-binding fragment thereof. Inpreferred embodiments, the TGFβ1-selective inhibitor is Ab42 or anantigen-binding fragment thereof.

Without being bound by theory, in some embodiments, sparing of TGFβinhibitors with anti-TGFβ3 activities may be especially useful fortreating patients who are diagnosed with a type of cancer known to behighly metastatic, myelofibrotic, and/or those having or are at risk ofdeveloping a fibrotic condition. Accordingly, the disclosure hereinincludes a TGFβ inhibitor for use in the treatment of cancer wherein theinhibitor does not inhibit TGFβ3 and wherein the patient has ametastatic cancer or myelofibrosis, or the patient has or is at risk ofdeveloping a fibrotic condition , wherein optionally the fibroticcondition is non-alcoholic steatohepatitis (NASH).

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has a TGFβ-related indication, a symptom of the indication,or a predisposition toward the indication, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affectthe indication, the symptom of the indication, or the predispositiontoward the indication.

Alleviating a TGFβ-related indication with an inhibitor, e.g., antibody,that selectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1complex includes delaying the development or progression of theindication, or reducing indication's severity. Alleviating theindication does not necessarily require curative results. As usedtherein, “delaying” the development of an indication associated with aTGFβ-related indication means to defer, hinder, slow, retard, stabilize,and/or postpone progression of the indication. This delay can be ofvarying lengths of time, depending on the history of the indicationand/or individuals being treated. A method that “delays” or alleviatesthe development of an indication, or delays the onset of the indication,is a method that reduces probability of developing one or more symptomsof the indication in a given time frame and/or reduces extent of thesymptoms in a given time frame, when compared to not using the method.Such comparisons are typically based on clinical studies, using a numberof subjects sufficient to give a statistically significant result.

Selection of a TGFβ Inhibitor for Treating a Fibrotic Disorder

Inhibitors of TGFβ include isoform-non-selective inhibitors andisoform-selective inhibitors, with the former being the majority ofknown TGFβ inhibitors/antagonists. Among the isoform-non-selectiveinhibitors are pan-inhibitors (TGFβ1 /2/3 inhibitors), TGFβ1/2inhibitors and TGFβ1/3 inhibitors. Isoform-selective inhibitors includeneutralizing antibodies that selectively bind one isoform and activationinhibitors that target latent proTGFβ complexes in an isoform-selectivemanner The class of activation inhibitors include context-independentand context-selecitve inhibitors. For example, isoform-specific,context-independent TGFβ1 inhibitors have also been described, whichbind pro/latent TGFβ1 presented by LTBP1/3, GARP, or LRRC33, and inhibitthe release of mature TGFβ1 from the presenting molecule complex (see,e.g., WO 2017/156500, WO 2020/014473 and WO 2020/014460). The entirecontents of each of the foregoing applications are incorporated hereinby reference. Context-specific antibodies that selectively bind aGARP-TGFβ1 complex and inhibit activation of TGFβ1 presented in thecontext of GARP have recently been described in WO 2018/013939.

The present invention includes specific inhibitors of ECM-associatedTGFβ1 activation, e.g., antibodies, and antigen-binding portionsthereof, that selectively bind a LTBP1/3-TGFβ1 complex, and whichinhibit activation of TGFβ1 presented in the context of LTBP1 or LTBP3.The present disclosure further provides guidance as to both selection ofa suitable TGFβ inhibitor among those listed above, tailored to certainpatient populations and related therapeutic regimen.

The surprising observations demonstrated herein (see Example 17; FIG.22) that showing that TGFβ3 inhibition may in fact aggravate ECMdysregulation are informative in a decision making process of selectingthe right TGFβ inhibitor. For example, for use in the treatment of afibrotic condition, it may be advantageous to select a TGFβ inhibitorthat lacks inhibitory activities towards TGFβ3. The observedexacerbation of fibrosis (e.g., profibrotic effects) in response toTGFβ3 inihbition raisesthe possibility that role of TGFβ3 expands beyondhomeostasis. Even more importantly, this may be relevant not only tofibrotic conditions but also in other disease contexts. Ample evidencesuggests that dysregulation of the ECM is found in a number of diseaseconditions, including fibrosis and cancer. Indeed, many of the keyprofibrotic genes are also recognized among markers of various cancers.These markers include, for example, collAl, col3A1, PAI-1, CCL2, ACTA2,FN-1, CTGF and TGFB1. Therefore, the finding that blockade of TGFβ3appears harmful in fibrosis (e.g., having a profibrotic effect) may beapplicable to a broader scope of conditions associated with ECMdysregulation.

The present invention encompasses insights into selecting “the rightTGFβ inhibitor” for “the right patient” to treat a disease conditionwith certain criteria and/or clinical features. In one aspect, thepresent invention provides use of preferred TGFβ1 inhibitors suitablefor a particular patient population with fibrotic conditions.Accordingly, the invention includes use of an LTBP1/LTBP3-proTGFβ1inhibitor in the treatment of a fibrotic condition in a subject, whereinthe subject benefits from immunosuppression. This is based on the notionthat at least a subset of TGFβ1 activities involves immune regulationwhich is mediated by GARP-associated and/or LRRC33-associated TGFβ1.Thus, the invention includes the recognition that use of TGFβ1inhibitors that also affect the immune aspect of TGFβ1 effects may bedetrimental for treating patients with fibrotic conditions whereimmunostimulation may cause exacerbation of the disease. The inventiontherefore aims at least in part to provide means of selectivelyinhibiting TGFβ1 effects within the ECM context (e.g., LTBP-associated)while sparing TGFβ1 effects associated with non-ECM contexts (e g ,immune cells, leukocytes, etc. expressing GARP or LRRC33 on cellsurface), so as to prevent unwanted immunostimulation. This approach maybe particularly advantageous in early-stage fibrosis, such asnoncirrhotic liver fibrosis.

In some embodiments, patient populations who benefit from both: i)inhibition of TGFβ1 signaling, and, ii) immunosuppression, include thosewho suffer from a severe or late stage organ fibrosis and who are toreceive an allograft organ transplant. The severe or late stage organfibrosis may be associated with IPF, CKD, and/or NASH. Such patients mayhave already received other therapies for treating the fibrotic disease,yet which may have failed to sufficiently treat or manage the condition.Attending physicians may determine that remaining treatment options mayinclude allograft transplantation. Such patients may be placed in a waitlist for an available organ for transplantation. Such patients may betreated with an immunosuppressant. A selective inhibitor ofLTBP1/LTBP3-presented TGFβ1 activation, which does not inhibitGARP-presented TGFβ1 activation, can be used to treat such patients,without raising risk of triggering immunostimulation mediated byeffector T cells. Similarly, following the transplantation, suchpatients may continue to receive the selective inhibitor ofLTBP1/LTBP3-presented TGFβ1 activation to avoid risk of an organrejection.

In some embodiments, patient populations who benefit from both: i)inhibition of TGFβ1 signaling, and, ii) immunosuppression, include thosewho suffer from a fibrotic disorder and who have an inflammatory orautoimmune condition.

In some embodiments, the patient or patient population has or is at riskof developing one or more autoimmune disorders, such as: Achalasia;Addison's disease; Adult Still's disease; Agammaglobulinemia; Alopeciaareata; Amyloidosis; Ankylosing spondylitis; Anti-GBM/Anti-TBMnephritis; Antiphospholipid syndrome; Autoimmune angioedema; Autoimmunedysautonomia; Autoimmune encephalomyelitis; Autoimmune hepatitis;Autoimmune inner ear disease (AIED); Autoimmune myocarditis; Autoimmuneoophoritis; Autoimmune orchitis; Autoimmune pancreatitis; Autoimmuneretinopathy; Autoimmune urticaria; Axonal & neuronal neuropathy (AMAN);Baló disease; Behcet's disease; Benign mucosal pemphigoid; Bullouspemphigoid; Castleman disease (CD); Celiac disease; Chagas disease;Chronic inflammatory demyelinating polyneuropathy (CIDP); Chronicrecurrent multifocal osteomyelitis (CRMO); Churg-Strauss Syndrome (CSS)or Eosinophilic Granulomatosis (EGPA); Cicatricial pemphigoid; Cogan'ssyndrome; Cold agglutinin disease; Congenital heart block; Coxsackiemyocarditis; CREST syndrome; Crohn's disease; Dermatitis herpetiformis;Dermatomyositis; Devic's disease (neuromyelitis optica); Discoid lupus;Dressler's syndrome; Endometriosis; Eosinophilic esophagitis (EoE);Eosinophilic fasciitis; Erythema nodosum; Essential mixedcryoglobulinemia; Evans syndrome; Fibromyalgia; Fibrosing alveolitis;Giant cell arteritis (temporal arteritis); Giant cell myocarditis;Glomerulonephritis; Goodpasture's syndrome; Granulomatosis withPolyangiitis; Graves' disease; Guillain-Barre syndrome; Hashimoto'sthyroiditis; Hemolytic anemia; Henoch-Schonlein purpura (HSP); Herpesgestationis or pemphigoid gestationis (PG); Hidradenitis Suppurativa(HS) (Acne Inversa); Hypogammalglobulinemia; IgA Nephropathy;IgG4-related sclerosing disease; Immune thrombocytopenic purpura (ITP);Inclusion body myositis (IBM); Interstitial cystitis (IC); Juvenilearthritis; Juvenile diabetes (Type 1 diabetes); Juvenile myositis (JM);Kawasaki disease; Lambert-Eaton syndrome; Leukocytoclastic vasculitis;Lichen planus; Lichen sclerosus; Ligneous conjunctivitis; Linear IgAdisease (LAD); Lupus; Lyme disease chronic; Meniere's disease;Microscopic polyangiitis (MPA); Mixed connective tissue disease (MCTD);Mooren's ulcer; Mucha-Habermann disease; Multifocal Motor Neuropathy(MMN) or MMNCB; Multiple sclerosis; Myasthenia gravis; Myositis;Narcolepsy; Neonatal Lupus; Neuromyelitis optica; Neutropenia; Ocularcicatricial pemphigoid; Optic neuritis; Palindromic rheumatism (PR);PANDAS; Paraneoplastic cerebellar degeneration (PCD); Paroxysmalnocturnal hemoglobinuria (PNH); Parry Romberg syndrome; Pars planitis(peripheral uveitis); Parsonage-Turner syndrome; Pemphigus; Peripheralneuropathy; Perivenous encephalomyelitis; Pernicious anemia (PA); POEMSsyndrome; Polyarteritis nodosa; Polyglandular syndromes type I, II, III;

Polymyalgia rheumatica; Polymyositis; Postmyocardial infarctionsyndrome; Postpericardiotomy syndrome; Primary biliary cirrhosis;Primary sclerosing cholangitis; Progesterone dermatitis; Psoriasis;Psoriatic arthritis; Pure red cell aplasia (PRCA); Pyoderma gangrenosum;Raynaud's phenomenon; Reactive Arthritis; Reflex sympathetic dystrophy;Relapsing polychondritis; Restless legs syndrome (RLS); Retroperitonealfibrosis; Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Schmidtsyndrome; Scleritis; Scleroderma; Sjogren's syndrome; Sperm & testicularautoimmunity; Stiff person syndrome (SPS); Subacute bacterialendocarditis (SBE); Susac's syndrome; Sympathetic ophthalmia (SO);Takayasu's arteritis; Temporal arteritis/Giant cell ;arteritisThrombocytopenic purpura (TTP); Tolosa-Hunt syndrome (THS);Transverse myelitis; Type 1 diabetes; Ulcerative colitis (UC);Undifferentiated connective tissue disease (UCTD); Uveitis; Vasculitis;Vitiligo; Vogt-Koyanagi-Harada Disease.

In some embodiments, the inflammatory or autoimmune condition isassociated with the fibrosis. Non-limiting examples of inflammatory orautoimmune conditions associated with fibrosis include musculardystrophy, such as DMD.

In other embodiments, where patient populations who benefit from both:i) inhibition of TGFβ1 signaling, and, ii) immunosuppression, includethose who suffer from a fibrotic disease and who have an inflammatory orautoimmune condition that is not directly associated with the fibrosis,but rather a discrete disorder.

Such inflammatory or autoimmune conditions, whether or not directlyassociated with the underlining fibrotic disease or separatecondition(s), may be caused by or associated with imbalance ofregulatory T cells (Treg) in human autoimmune diseases. For example,such disorders that are linked to Treg dysregulation include, but arenot limited to: Juvenile idiopathic arthritis; Rheumatoid arthritis(RA); Spondyloarthritis; Psoriatic arthritis; HCV mixedcryoglobulinaemia; cryoglobulinaemia; Multiple sclerosis; Autoimmuneliver disease; Systemic lupus erythematodes; Immune-mediated diabetes;Myasthenia gravis; Primary Sjogren syndrome; Kawasaki disease; and,Inflammatory bowel disease (IBD).

Thus, LTBP1/3-sepective inhibitors of TGFβ1 signaling, such as thosedescribed herein,can be used to treat patients who suffer from afibrotic condition and inflammatory or autoimmune condition such as oneor more of the disorders listed above. The LTBP1/3-sepective inhibitorsof TGFβ1 signaling used accordingly can treat or alleviateTGFβ1-dependent fibrosis in the ECM, while sparing immune-associatedTGFβ1 signaling.

Accordingly, related methods of the invention include methods forselecting an appropriate TGFβ1 inhibitor for treating a fibroticdisorder, based on the clinical manifestations of the fibrotic disorderin a subject. In one embodiment, the invention provides a method ofselecting an isoform-specific TGFβ1 inhibitor for treatment of afibrotic disorder in a subject. The method comprises (a) determiningwhether the fibrotic disorder manifests clinical presentations includingfibrosis and one or more of inflammation, immune suppression,proliferative dysregulation, and need for an allograft transplant, and(b) selecting an isoform-specific, context-dependent TGFβ1 inhibitor oran isoform-specific, context-independent TGFβ1 inhibitor for treatmentof the fibrotic disorder based on the clinical presentations determinedin step (a). In another embodiment, the invention provides a method oftreating a subject having a fibrotic disorder, comprising selecting atreatment regimen including an isoform-specific TGFβ1 inhibitor for thesubject, and administering the selected treatment regimen to thesubject, wherein the selection comprises (a) determining whether thefibrotic disorder manifests clinical presentations including fibrosisand one or more of the following: inflammation, immune suppression,proliferative dysregulation, and need for an allograft transplant; and(b) selecting a treatment regimen comprising an isoform-specific,context-dependent TGFβ1 inhibitor or an isoform-specific,context-independent TGFβ1 inhibitor, based on the clinical presentationsdetermined in step (a).

Subjects afflicted with fibrotic disorders can display a wide range ofsymptoms, in addition to fibrosis. The specific combination of clinicalmanifestations in a subject can guide the selection of an appropriateTGFβ1-inhibitory treatment regimen. For example, a context-independent,isoform-specific TGFβ1 inhibitor can be used to treat the subject if thesubject's clinical manifestations indicate a need for inhibition ofTGFβ1, without modulating the activity of TGFβ2 or TGFβ3. A treatmentregimen including a LTBP context-specific inhibitor can be used to treatthe subject if the subject's clinical manifestations indicate thatinhibition of TGFβ1 in the extracellular matrix would be beneficial. ALTBP context-specific inhibitor is also advantageous if the subject'sclinical manifestations indicate that stimulation of immune effectorcells is undesirable. A GARP context-specific inhibitor can be used totreat the subject if the subject's clinical manifestations indicate thatblocking the activation/release of TGFβ1 on regulatory T cells (Tregcells) would be beneficial, e.g., to prevent Treg cells from suppressingeffector T cell activity. A LRRC33 context-specific inhibitor can beused to treat the subject if the subject's clinical manifestationsindicate that blocking the activation/release of TGFβ1 on myeloid cells,monocytes, macrophages, dendritic cells and/or microglia would bebeneficial, e.g., to reverse or reduce immune suppression in thesubject.

By way of example, a subject having a fibrotic disorder may displayclinical manifestations including fibrosis, inflammation, immunesuppression, and proliferative dysregulation. Fibrotic disorders whichcommonly present with the foregoing combination of symptoms include,e.g., myelofibrosis. In this embodiment, an isoform-specific,context-independent TGFβ1 inhibitor can be selected for treating thesubject.

A subject having a fibrotic disorder may display clinical manifestationsincluding fibrosis, inflammation, and need for an allograft transplant.Fibrotic disorders which commonly present with the foregoing combinationof symptoms include, e.g., organ fibrosis, such as kidney fibrosis(e.g., fibrosis associated with chronic kidney disease), liver fibrosis(e.g., fibrosis associated with nonalcoholic steatohepatitis (NASH)), orlung fibrosis (e.g., fibrosis associated with idiopathic pulmonaryfibrosis (IPF)). In this embodiment, a context-specific LTBP1/3-specificinhibitor is selected for treating the subject.

In another example, a subject having a fibrotic disorder may displayclinical manifestations including fibrosis and inflammation. Fibroticdisorders which commonly present with the foregoing combination ofsymptoms include, e.g., scleroderma. In this embodiment, acontext-specific LTBP1/3-specific inhibitor is selected for treating thesubject. Additional fibrotic disorders which commonly present with theforegoing combination of symptoms include, e.g., degenerative diseases,such as muscular dystrophy, e.g., Duchenne muscular dystrophy (DMD). Inthis embodiment, a context-specific LTBP1/3-specific inhibitor isselected for treating the subject.

A subject having a fibrotic disorder may display clinical manifestationsincluding immune suppression and proliferative dysregulation. Fibroticdisorders which commonly present with the foregoing combination ofsymptoms include, e.g., solid tumors. In some embodiments, the solidtumor is a malignant tumor. In other embodiments, the solid tumor is abenign tumor. In an exemplary embodiment, the subject has desmoplasia(e.g., pancreatic desmoplasia). In some embodiments, patients may have asolid tumor that has been assessed as “inoperable” or not suitable forsurgical resection. Thus, in some embodiments, patients are notcandidates for surgical resection of the tumor. However, TGFβ1inhibition therapy comprising a context-selective TGFβ1 inhibitor of thepresent invention may reverse such non-candidate patients to be moresuited for receiving a surgery. In some embodiments, subjects having asolid tumor are poorly responsive to cancer therapy (e.g., the tumor isresistant to the cancer therapy), such as chemotherapy, radiationtherapy, CAR-T therapy and checkpoint inhibitor therapy. TGFβ1inhibition therapy comprising a context-selective TGFβ1 inhibitor of thepresent invention may at least in part reverse the resistance to renderthe patient more responsive to the cancer therapy. In some embodiments,a combination therapy comprising both the context-selective TGFβ1inhibition therapy and the cancer therapy may synergistically treat thecancer. In some embodiments, the context-selective TGFβ1 inhibitiontherapy administered in conjunction with the cancer therapy may reducethe required dosage of the cancer therapy to produce equivalent orimproved clinical effects.

In another exemplary embodiment, the subject has fibroids. In theforegoing embodiments, in which the fibrotic disorder displays clinicalmanifestations including immune suppression and proliferativedysregulation, a context-specific LTBP1/3-specific inhibitor and/or acontext-specific GARP-specific inhibitor are selected for treating thesubject.

In another aspect, the invention provides a method of treating a subjecthaving a fibrotic disorder with an isoform-specific, LTBP1/3context-specific TGFβ1 inhibitor, by selecting a subject phaving afibrotic disorder manifesting clinical presentations including fibrosisand the need for an allograft transplant, and administering an effectiveamount of an isoform-specific, LTBP1/3-specific TGFβ1 inhibitor to thesubject. In one embodiment, the method comprises determining whether thefibrotic disorder manifests clinical presentations including fibrosisand the need for an allograft transplant. The LTBP1/3-specific TGFβ1inhibitor is administered to the subject if the subject exhibitssymptoms including fibrosis and the need for an allograft transplant.

In another aspect, the invention provides a method of treating a subjecthaving a fibrotic disorder with an isoform-specific, context-independentTGFβ1 inhibitor, by selecting a subject having a fibrotic disordermanifesting clinical presentations including fibrosis, immunesuppression, and/or proliferative dysregulation, and administering aneffective amount of an isoform-specific, context-independent TGFβ1inhibitor to the subject. In one embodiment, the method comprisesdetermining whether the fibrotic disorder manifests clinicalpresentations including fibrosis, immune suppression, and/orproliferative dysregulation. The isoform-specific, context-independentTGFβ1 inhibitor is administered to the subject if the subject inhibitssymptoms including fibrosis, immune suppression, and/or proliferativedysregulation.

Clinical manifestations including inflammation, immune suppression,proliferative dysregulation, and/or the need for an allograft transplantcan be determined in a subject having a fibrotic disorder using methodsand practices known in the art. Such methods include, for example,physical examination and standard diagnostic tests. In one embodiment,inflammation can be assessed by determining if a subject displays anelevated level of inflammatory biomarkers in plasma, blood, or serum.Such inflammatory biomarkers include, for example, C-reactive protein,interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor a(TNF-α), or combinations thereof. Blood tests including erythrocytesedimentation rate (ESR) and plasma viscosity (PV) can also indicate thepresence of inflammation in a subject with a fibrotic disorder. Inanother embodiment, immune suppression can be assessed by determiningthe number and composition of a subject's blood cells, e.g., T cells, Bcells, NK cells, monocytes, macrophages, etc Immune suppression can alsobe assessed by determining if the subject is taking or has a history oftaking immunosuppressant medications, or determining if the subject hasa condition associated with immune suppression (e.g., hematologicalmalignancies, HIV/AIDS, etc.). In another embodiment, proliferativedysregulation can be assessed using standard tests including bloodtests, biopsy, and/or imaging procedures such as CT scan, ultrasound,and MRI. Other standard tests for diagnosing cancer (e.g., biomarkertests, etc.) can also be used to assess proliferative dysregulation. Theneed for an allograft transplant can be determined by a clinician usingstandard procedures. In one embodiment, the loss or partial loss oforgan function, or an increased likelihood of loss of organ function,indicates the need for a transplant.

As mentioned, the present invention provides selective targeting of theECM-associated TGFβ1 complexes enabled by the use of antibodies that arecapable of specifically binding LTBP-presented TGFβ1 precursors. Whilesome antibodies of the present invention are capable of binding andinhibiting both LTBP1- and LTBP3-associated proTGFβ1 complexes, othersshow even greater selectivity in that they only bind eitherLTBP1-proTGFβ1 or LTBP3-proTGFβ1.

The invention therefore encompasses the recognition that certain patientpopulations may benefit from TGFβ1 inhibition therapy comprising acontext-selective inhibitor that is specific to LTBP1/3-proTGFβ1, overTGFβ inhibitors that also affect the immune components of TGFβ signalingnamely, TGFβ associated with GARP. Accordingly, it is contemplatedherein that to treat a TGFβ-related condition in a subject who has or isat risk of developing an autoimmune condition, a TGFβ inhibitor thatselectively inhibits matrix-associated TGFβ (such as LTBP1/3context-selective inhibitors of TGFβ1 disclosed herein) may providetherapeutic benefits while minimizing risk of overstimulating the immunesystem. Such subject may suffer from or may be at risk of developing anautoimmune disorder, such as: Achalasia; Addison's disease; AdultStill's disease; Agammaglobulinemia; Alopecia areata; Amyloidosis;Ankylosing spondylitis; Anti-GBM/Anti-TBM nephritis; Antiphospholipidsyndrome; Autoimmune angioedema; Autoimmune dysautonomia; Autoimmuneencephalomyelitis; Autoimmune hepatitis; Autoimmune inner ear disease(AIED); Autoimmune myocarditis; Autoimmune oophoritis; Autoimmuneorchitis; Autoimmune pancreatitis; Autoimmune retinopathy; Autoimmuneurticaria; Axonal & neuronal neuropathy (AMAN); Baló disease; Behcet'sdisease; Benign mucosal pemphigoid; Bullous pemphigoid; Castlemandisease (CD); Celiac disease; Chagas disease; Chronic inflammatorydemyelinating polyneuropathy (CIDP); Chronic recurrent multifocalosteomyelitis (CRMO); Churg-Strauss Syndrome (CSS) or EosinophilicGranulomatosis (EGPA); Cicatricial pemphigoid; Cogan's syndrome; Coldagglutinin disease; Congenital heart block; Coxsackie myocarditis; CRESTsyndrome; Crohn's disease; Dermatitis herpetiformis; Dermatomyositis;Devic's disease (neuromyelitis optica); Discoid lupus; Dressler'ssyndrome; Endometriosis; Eosinophilic esophagitis (EoE); Eosinophilicfasciitis; Erythema nodosum; Essential mixed cryoglobulinemia; Evanssyndrome; Fibromyalgia; Fibrosing alveolitis; Giant cell arteritis(temporal arteritis); Giant cell myocarditis; Glomerulonephritis;Goodpasture's syndrome; Granulomatosis with Polyangiitis; Graves'disease; Guillain-Barre syndrome; Hashimoto's thyroiditis; Hemolyticanemia; Henoch-Schonlein purpura (HSP); Herpes gestationis or pemphigoidgestationis (PG); Hidradenitis Suppurativa (HS) (Acne Inversa);Hypogammalglobulinemia; IgA Nephropathy; IgG4-related sclerosingdisease; Immune thrombocytopenic purpura (ITP); Inclusion body myositis(IBM); Interstitial cystitis (IC); Juvenile arthritis; Juvenile diabetes(Type 1 diabetes); Juvenile myositis (JM); Kawasaki disease;Lambert-Eaton syndrome; Leukocytoclastic vasculitis; Lichen planus;Lichen sclerosus; Ligneous conjunctivitis; Linear IgA disease (LAD);Lupus; Lyme disease chronic; Meniere's disease; Microscopic polyangiitis(MPA); Mixed connective tissue disease (MCTD); Mooren's ulcer;Mucha-Habermann disease; Multifocal Motor Neuropathy (MMN) or MMNCB;Multiple sclerosis; Myasthenia gravis; Myositis; Narcolepsy; NeonatalLupus; Neuromyelitis optica; Neutropenia; Ocular cicatricial pemphigoid;Optic neuritis; Palindromic rheumatism (PR); PANDAS; Paraneoplasticcerebellar degeneration (PCD); Paroxysmal nocturnal hemoglobinuria(PNH); Parry Romberg syndrome; Pars planitis (peripheral uveitis);Parsonage-Turner syndrome; Pemphigus; Peripheral neuropathy; Perivenousencephalomyelitis; Pernicious anemia (PA); POEMS syndrome; Polyarteritisnodosa; Polyglandular syndromes type I, II, III; Polymyalgia rheumatica;Polymyositis; Postmyocardial infarction syndrome; Postpericardiotomysyndrome; Primary biliary cirrhosis; Primary sclerosing cholangitis;Progesterone dermatitis; Psoriasis; Psoriatic arthritis; Pure red cellaplasia (PRCA); Pyoderma gangrenosum; Raynaud's phenomenon; ReactiveArthritis; Reflex sympathetic dystrophy; Relapsing polychondritis;Restless legs syndrome (RLS); Retroperitoneal fibrosis; Rheumatic fever;Rheumatoid arthritis; Sarcoidosis; Schmidt syndrome; Scleritis;Scleroderma; Sjogren's syndrome; Sperm & testicular autoimmunity; Stiffperson syndrome (SPS); Subacute bacterial endocarditis (SBE); Susac'ssyndrome; Sympathetic ophthalmia (SO); Takayasu's arteritis; Temporalarteritis/Giant cell ; arteritisThrombocytopenic purpura (TTP);Tolosa-Hunt syndrome (THS); Transverse myelitis; Type 1 diabetes;Ulcerative colitis (UC); Undifferentiated connective tissue disease(UCTD); Uveitis; Vasculitis; Vitiligo; Vogt-Koyanagi-Harada Disease.

LTBP1 and LTBP3 are both components of the ECM, where they can displayor “present” a latent TGFβ precursor complex. Some observations fromexpression studies raise the possibility that deletion, ablation orfunctional inhibition of LTBP3 may cause certain toxicities. LTBP3−/−mice (as well as some human mutations) have short stature, as well asbone and dental anomalies These phenotypes are likely associated withdisruptions in development, however, but it is possible that LTBP3 playsa role in homeostasis of these tissues in adults (expression in adultbone is reported). Based on these observations, in certain clinicalsituations (where the disease manifests in a tissue known to expressLTBP3 and associated with toxicities) or in certain patient populations,such as pediatric patients who are still in active development, it maybe advisable to avoid potential toxicities of LTBP3-related inhibition.Loss of LTBP1 function does appear to be sufficient to protect againstat least some forms of fibrosis, as LTBP1 −/− KO mice are protectedagainst liver fibrosis (induced by bile duct ligation). Taken together,these data raise the possibility that LTBP1-specific TGFβ1 inhibitioncould have a superior safety profile as compared to LTBP1/3-TGFβ1inhibitors in certain situations.

Accordingly, selection of patitents or patient poplulations suitable orlikely to benefit from the LTBP1/3-selective inhibitors of the presentinvention may involve evaluating or confirming expression profiles ofLTBP1, LTBP2, LTBP3, LTBP4, GARP, LRRC33, pro- or mature TGFβ1, pro- ormature TGFβ2, pro- or mature TGFβ3, or any combinations thereof.Expression profiles may be obtained by measuring the presence/absence orlevels of mRNA and/or proteins in suitable assays from biologicalsamples collected from the subject (e.g., patients). In someembodicments, a sluble circulating fragment(s) of LAP may be used assurrogate marker for the expression of the particular TGFβ3 isoform. Insome embodiments, TGFβ1 LAP fragments may be used as a marker offibrogenesis. See for example, US patent 8,198,412, the contents ofwhich are incorporated herein by reference.

Genetic Markers of Disease:

It has been observed that abnormal activation of the TGFβ1 signaltransduction pathway in various disease conditions is associated withaltered gene expression of a number of markers. These gene expressionmarkers (e.g., as measured by mRNA) include, but are not limited to:Serpine 1 (encoding PAI-1), MCP-1 (also known as CCL2), Collal, Col3a1,FN1, TGFβ1, CTGF, ACTA2 (encoding a-SMA), SNAI1 (drives EMT in fibrosisand metastasis by downregulating E-cadherin (Cdhl), MMP2 (matrixmetalloprotease associated with EMT), MMP9 (matrix metalloproteaseassociated with EMT), TIMP1 (matrix metalloprotease associated withEMT), FOXP3 (marker of Treg induction), CDH1 (E cadherin (marker ofepithelial cells) which is downregulated by TGFβ3), and, CDH2 (Ncadherin (marker of mesenchymal cells) which is upregulated by TGFβ3).Interestingly, many of these genes are implicated to play a role in adiverse set of disease conditions, including various types of organfibrosis, as well as in many cancers, which include myelofibrosis.Indeed, pathophysiological link between fibrotic conditions and abnormalcell proliferation, tumorigenesis and metastasis has been suggested. Seefor example, Cox and Erler (2014) Clinical Cancer Research 20(14):3637-43 “Molecular pathways: connecting fibrosis and solid tumormetastasis”; Shiga et al. (2015) Cancers 7:2443-2458 “Cancer-associatedfibroblasts: their characteristics and their roles in tumor growth”;Wynn and Barron (2010) Semin. Liver Dis. 30(3): 245-257 “Macrophages:master regulators of inflammation and fibrosis”, contents of which areincorporated herein by reference. Without wishing to be bound by aparticular theory, the inventors of the present disclosure contemplatethat the TGFβ1 signaling pathway may in fact be a key link between thesebroad pathologies.

The ability of chemotactic cytokines (or chemokines) to mediateleukocyte recruitment (e.g., monocytes/macrophages) to injured ordisease tissues has crucial consequences in disease progression. Membersof the C-C chemokine family, such as monocyte chemoattractant protein 1(MCP-1), also known as CCL2, macrophage inflammatory protein 1-alpha(MIP-1 a), also known as CCL3, and MIP-113, also known as CCL4, havebeen implicated in this process.

For example, MCP-1/CCL2 is thought to play a role in both fibrosis andcancer. MCP-1/CCL2 is characterized as a profibrotic chemokine and is amonocyte chemoattractant, and evidence suggests that it may be involvedin both initiation and progression of cancer. In fibrosis, MCP-1/CCL2has been shown to play an important role in the inflammatory phase offibrosis. For example, neutralization of MCP-1 resulted in a dramaticdecrease in glomerular crescent formation and deposition of type Icollagen. Similarly, passive immunotherapy with either anti-MCP-1 oranti-MIP-1 alpha antibodies is shown to significantly reduce mononuclearphagocyte accumulation in bleomycin-challenged mice, suggesting thatMIP-1 alpha and MCP-1 contribute to the recruitment of leukocytes duringthe pulmonary inflammatory response (Smith, Biol Signals. 1996Jul.-Aug.; 5(4):223-31, “Chemotactic cytokines mediate leukocyterecruitment in fibrotic lung disease”). Elevated levels of MIP-lalpha inpatients with cystic fibrosis and multiple myeloma have been reported(see, for example: Mrugacz et al., J Interferon Cytokine Res. 2007 Jun;27(6):491-5), supporting the notion that MIP-la is associated withlocalized or systemic inflammatory responses.

Lines of evidence point the involvement of C-C chemokines in tumorprogression. For example, tumor-derived MCP-1/CCL2 can promote“pro-cancer” phenotypes in macrophages. For example, in lung cancer,MCP-1/CCL2 has been shown to be produced by stromal cells and promotemetastasis. In human pancreatic cancer, tumors secrete CCL2, andimmunosuppressive CCR2-positive macrophages infiltrate these tumors.Patients with tumors that exhibit high CCL2 expression/low CD8 T-cellinfiltrate have significantly decreased survival. Without wishing to bebound by particular theory, it is contemplated that monocytes that arerecruited to an injuried or diseased tissue environment may subsequentlybecome polarized in response to local cues (such as in response totumor-derived cytokines), therby further contributing to diseaseprogression. These M2-like macrophages are likely to contribute toimmune evasion by suppressing effector cells, such as CD4+and CD8+Tcells. In some embodiments, this process is in part mediated byLRRC33-TGFβ1 expressed by activated macrophages. In some embodiments,the process is in part mediated by GARP-TGFβ1 expressed by Tregs.

Similarly, involvement of PAI-1/Serpinel has been implicated in avariety of cancers, angiogenesis, inflammation, neurodegenerativediseases (e.g., Alzheimer's Disease). Elevated expression of PAI-1 intumor and/or serum is correlated with poor prognosis (e.g., shortersurvival, increased metastasis) in various cancers, such as breastcancer and bladder cancer (e.g., transitional cell carcinoma) as well asmyelofibrosis. In the context of fibrotic conditions, PAI-1 has beenrecognized as an important downstream effector of TGFβ1-inducedfibrosis, and increased PAI-1 expression has been observed in variousforms of tissue fibrosis, including lung fibrosis (such as IdiopathicPulmonary Fibrosis (IPF)), kidney fibrosis, liver fibrosis andscleroderma. In some embodiments, the process is in part mediated byECM-associated TGFβ1, e.g., via LTBP1 and/or LTBP3.

Accordingly, in some embodiments, in vivo effects of the TGFβ1 inhibitortherapy may be assessed by measuring changes in gene markers. Suitablemarkers include TGFβ (e.g., TGFβ31, TGFβ2, and TGFβ3 ). Suitable markersmay also include one or more presenting molecules for TGFβ1 (e.g.,TGFβ31, TGFβ2, and TGFβ3 ), such as LTBP1, LTBP3, GARP (or LRRC32) andLRRC33. In some embodiments, suitable markers include mesenchymaltransition genes (e.g., AXL, ROR2, WNT5A, LOXL2, TWIST2, TAGLN, and/orFAP), immunosuppressive genes (e.g., IL10, VEGFA, VEGFC), monocyte andmacrophage chemotactic genes (e.g., CCL2, CCL3, CCL4, CCL7, CCL8 andCCL13), and/or various fibrotic markers discussed herein. Preferredmarkers are plasma markers.

In some embodiments, an LTBP complex inhibitor of TGFβ1 is used in thetreatment of a disease associated with overexpression of one or more ofthe following: PAI-1 (encoded by Serpinel), MMP2, MMP9, MCP-1 (alsoknown as CCL2), Collal, Col3al, FN1, TGFβ1, CTGF, a-SMA, ITGA11, andACTA2, wherein the treatment comprises administration of the inhibitorto a subject suffering from the disease in an amount effective to treatthe disease. In some embodiments, the inhibitor is used to treat adisease associated with overexpression of PAI-1, MCP-1/CCL2, CTGF,and/or a-SMA. In some embodiments, the disease is myelofibrosis. In someembodiments, the disease is cancer, for example, cancer comprising asolid tumor. In some embodiments, the disease is organ fibrosis, e.g.,fibrosis of the liver, the kidney, the lung, the muscle, the skin and/orthe cardiac or cardiovascular tissue. In some embodiments, the diseaseis Alport Syndrom. In some embodiments, the inhibitor reduces expressionof one or more of the following: PAI-1 (encoded by Serpinel), MMP2,MMP9, MCP-1 (also known as CCL2), Collal, Col3al, FN1, TGFβ1, CTGF,a-SMA, ITGA11, and ACTA2.

Another biomarker which may be used to assess the in vivo effects of theTGFβ1 inhibitor therapy is blood urea nitrogen (BUN). Urea is naturallyformed in the body as a by-product of protein breakdown. The ureatravels from you liver to your kidneys where it is filtered/removed fromthe blood. Accordingly, BUN levels may increase in situations when apatient's kidneys are not functioning properly. For example, patientshaving kidney fibrosis may display increased BUN. Accordingly, in someembodiments, BUN is measured to assess the in vivo effects of theLTBP-specific inhibitors of TGFβ1 as described herein. In otherembodiments, an LTBP-specific inhibitor of TGFβ1 is used in thetreatment of a disease associated with increased BUN (e.g., kidneyfibrosis and/or acute or chronic kidney disease, damage, or failure). Ina particular embodiment, the disease associated with increased BUN isAlport Syndrome.

Accordingly, the present disclosure includes a method of selecting acandidate patient or patient population likely to respond to a TGFβ1inhibition therapy. Such method may comprise a step of testing abiological sample collected from the patient (or patient population),such as biopsy samples, for the expression of one or more of the markersdiscussed herein. Similarly, such genetic marker(s) may be used forpurposes of monitoring the patient's responsiveness to a therapy.Monitoring may include testing two or more biological samples collectedfrom the patient, for example, before and after administration of atherapy, and during the course of a therapeutic regimen over time, toevaluate changes in gene expression levels of one or more of themarkers, indicative of therapeutic response or effectiveness.

In some embodiments, a method of selecting a candidate patient orpatient population likely to respond to a TGFβ1 inhibition therapy maycomprise a step of identifying a patient or patient populationpreviously tested for the genetic marker(s), such as those describedherein, which showed aberrant expression thereof. In some embodiments,the aberrant marker expression includes elevated levels of at least oneof the following: TGFβ1 (and/or TGFB1), LRRC33, GARP, LTBP1, LTBP3,CCL2, CCL3, PAI-1/Serpinel, MMP2, MMP9, Col1a1, Col3a1, FN1, CTGF,α-SMA, ITGAll, and ACTA2. In some embodiments, the patient or patientpopulation (e.g., biological samples collected therefrom) shows elevatedTGFβ1 activation, phospho-Smad2/3, or combination thereof. In someembodiments, the patient or patient population shows elevated BUN.

Combination Therapies

The disclosure further encompasses pharmaceutical compositions andrelated methods used as combination therapies for treating subjects whomay benefit from TGFβ1 inhibition in vivo. In any of these embodiments,such subjects may receive combination therapies that include a firstcomposition comprising at least one TGFβ1 inhibitor, e.g., antibody orantigen-binding portion thereof, described herein, in conjunction with asecond composition comprising at least one additional therapeuticintended to treat the same or overlapping disease or clinical condition.The first and second compositions may both act on the same cellulartarget, or discrete cellular targets. In some embodiments, the first andsecond compositions may treat or alleviate the same or overlapping setof symptoms or aspects of a disease or clinical condition. In someembodiments, the first and second compositions may treat or alleviate aseparate set of symptoms or aspects of a disease or clinical condition.To give but one example, the first composition may treat a disease orcondition associated with TGFβ1 signaling, while the second compositionmay treat inflammation or fibrosis associated with the same disease,etc. Such combination therapies may be administered in conjunction witheach other. The phrase “in conjunction with,” in the context ofcombination therapies, means that therapeutic effects of a first therapyoverlaps temporarily and/or spatially with therapeutic effects of asecond therapy in the subject receiving the combination therapy. Thus,the combination therapies may be formulated as a single formulation forconcurrent administration, or as separate formulations, for sequentialadministration of the therapies.

In preferred embodiments, combination therapies produce synergisticeffects in the treatment of a disease. The term “synergistic” refers toeffects that are greater than additive effects (e.g., greater efficacy)of each monotherapy in aggregate.

In some embodiments, combination therapies comprising a pharmaceuticalcomposition described herein produce efficacy that is overall equivalentto that produced by another therapy (such as monotherapy of a secondagent) but are associated with fewer unwanted adverse effect or lesssevere toxicity associated with the second agent, as compared to themonotherapy of the second agent. In some embodiments, such combinationtherapies allow lower dosage of the second agent but maintain overallefficacy. Such combination therapies may be particularly suitable forpatient populations where a long-term treatment is warranted and/orinvolving pediatric patients.

Accordingly, the invention provides pharmaceutical compositions andmethods for use in combination therapies for the reduction of TGFβ1protein activation and the treatment or prevention of diseases orconditions associated with TGFβ1 signaling, as described herein.Accordingly, the methods or the pharmaceutical compositions furthercomprise a second therapy. In some embodiments, the second therapy maybe useful in treating or preventing diseases or conditions associatedwith TGFβ1 signaling. The second therapy may diminish or treat at leastone symptom(s) associated with the targeted disease. The first andsecond therapies may exert their biological effects by similar orunrelated mechanisms of action; or either one or both of the first andsecond therapies may exert their biological effects by a multiplicity ofmechanisms of action.

It should be understood that the pharmaceutical compositions describedherein may have the first and second therapies in the samepharmaceutically acceptable carrier or in a different pharmaceuticallyacceptable carrier for each described embodiment. It further should beunderstood that the first and second therapies may be administeredsimultaneously or sequentially within described embodiments.

In one embodiment, the inhibitors, e.g., antibodies, described hereinthat selectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1complex can be administered with another agent that inhibits TGFβ1activity. For example, the second agent can be another context-specificTGFβ1 inhibitor. In one embodiment, the combination therapy comprises(i) an inhibitor, e.g., antibody or antigen-binding portion thereof,that selectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1complex, and (ii) an inhibitor, e.g., antibody or antigen-bindingportion thereof, that selectively binds a GARP-TGFβ1 complex. In anotherembodiment, the combination therapy comprises (i) an inhibitor, e.g.,antibody or antigen-binding portion thereof, that selectively binds aLTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, and (ii) an inhibitor,e.g., antibody or antigen-binding portion thereof, that selectivelybinds a LRRC33-TGFβ1 complex. Context-specific antibodies thatselectively bind LRRC33-TGFβ1 are described, for example, in US62/503,785, and context-specific antibodies that selectively bindGARP-TGFβ1 are described, above. The entire contents of the foregoingapplications are incorporated by reference herein. In one embodiment,the combination therapy comprises (i) an inhibitor, e.g., antibody orantigen-binding portion thereof, that selectively binds a LTBP1-TGFβ1complex and/or a LTBP3-TGFβ1 complex, and (ii) an context-independentinhibitor, e.g., antibody or antigen-binding portion thereof, thatselectively binds pro/latent TGFβ1 in a complex with a presentingmolecule (e.g., LTBP1/3, GARP, and/or LRRC33). Context-independentinhibitors of TGFβ1 are described, for example, in WO 2017/156500, theentire contents of which are incorporated herein by reference.

The one or more anti-TGFβ1 inhibitors, e.g., antibodies, orantigen-binding portions thereof, of the invention may be used incombination with one or more additional therapeutic agents. Examples ofthe additional therapeutic agents which can be used with an anti-TGFβantibody of the invention include, but are not limited to, a myostatininhibitor, a VEGF agonist, an IGF1 agonist, an FXR agonist, a CCR2inhibitor, a CCR5 inhibitor, a dual CCR2/CCR5 inhibitor, a lysyloxidase-like-2 inhibitor, an ASK1 inhibitor, an Acetyl-CoA Carboxylase(ACC) inhibitor, a p38 kinase inhibitor, Pirfenidone, Nintedanib,selonsertib, cilofexor, firsocostat, Pirfenidone, obeticholic acid,elafibranor,an anti-CD147 antibody, an anti-GP73 antibody, a Galactin-1inhibitor, selonsertib, a caspase inhibitor (Emricasan, IDN-6556,PF-03491390), a GDF11 inhibitor, a GDF8/myostatin inhibitor, and thelike. The GDF8/myostatin inhibitor is preferably a myostatin-selectiveinhibitor (e.g., an antibody or antigen-biding fragment). Themyostatin-selective inhibitor may bind latent myostatin. Non-limitingexamples of myostatin-selective inhibitors include SRK-015 (e.g., seeWO2017/218592A1) and trevogrumab, or any variant thereof, or an antibodyaccording to WO 2016/098357.

In some embodiments, the additional agent is a checkpoint inhibitor. Insome embodiments, the additional agent is selected from the groupconsisting of a PD-1 antagonist, a PDL1 antagonist, a PD-L1 or PDL2fusion protein, a CTLA4 antagonist, a GITR agonist, an anti-ICOSantibody, an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-0X₄₀antibody, an anti-CD27 antibody, an anti-CD70 antibody, an anti-CD47antibody, an anti-41BB antibody, an anti-PD-1 antibody, an oncolyticvirus, and a PARP inhibitor. In some embodiments, the additional therapyis radiation. In some embodiments, the additional agent is achemotherapeutic agent. In some embodiments, the chemotherapeutic agentis Taxol. In some embodiments, the additional agent is ananti-inflammatory agent. In some embodiments, the additional agentinhibits the process of monocyte/macrophage recruitment and/or tissueinfiltration. In some embodiments, the additional agent is an inhibitorof hepatic stellate cell activation. In some embodiments, the additionalagent is a chemokine receptor antagonist, e.g., CCR2 antagonists andCCR5 antagonists. In some embodiments, such chemokine receptorantagonist is a dual specific antagonist, such as a CCR2/CCR5antagonist. In some embodiments, the additional agent to be administeredas combination therapy is or comprises a member of the TGFβ superfamilyof growth factors or regulators thereof. In some embodiments, such agentis selected from modulators (e.g., inhibitors and activators) ofGDF8/myostatin and GDF11. In some embodiments, such agent is aninhibitor of GDF8/myostatin signaling. In some embodiments, such agentis a monoclonal antibody that binds a pro/latent myostatin complex andblocks activation of myostatin. In some embodiments, the monoclonalantibody that binds a pro/latent myostatin complex and blocks activationof myostatin does not bind free, mature myostatin.

Combination therapy that includes a TGFβ inhibitor (such asTGFβ1-selective inhibitors disclosed herein), in conjunction with one ormore additional therapies, may be considered for treating a variety ofliver diseases. Non-limiting examples of liver diseases include:non-alcoholic fatty liver disease (NAFLD), e.g., non-alcoholic fattyliver (NAFL) and non-alcoholic steatohepatitis (NASH), which mayinclude: noncirrhotic NASH with liver fibrosis, liver cirrhosis, NASHwith compensated cirrhosis, NASH with decompensated cirrhosis, liverinflammation with fibrosis, liver inflammation without fibrosis; stage 2and 3 liver fibrosis, stage 4 fibrosis (NASH cirrhosis or cirrhotic NASHwith fibrosis), primary biliary cholangitis (PBC) (formerly known asprimary biliary cirrhosis), and primary sclerosing cholangitis (PSC).

One or more of the following therapies may be used in conjunction withthe TGFβ inhibitor (such as TGFβ1-selective inhibitors disclosed herein)for the treatment of a liver disease such as those listed above:Pioglitazone (PPARy agonist); Elafibranor (PPARα/β agonist);Saroglitazar (PPARα/γ agonist); Obeticholic acid (FXR agonist);Liraglutide (GLP-1 receptor agonist); Aramchol (SCD inhibitor);Volixibat (SHP-626) (ASBT inhibitor); BMS-986036 (FGF-21 analogue);NGM-282 (FGF-19 analogue); Tesamorelin (GHRH analogue); NDI-010976 (ACCinhibitor); GS-9674 (FXR agonist); Dur-928 (Sulfated oxysterol); AZD4076(miR-103/107 antagonist); Rosuvastatin (HMG-CoA reductase inhibitor);INT-767 (FXR/TGR5 agonist); Sevelamer (Bile acid sequestrant); Vitamin E(Antioxidant); Pentoxifylline (PDE inhibitor); Cenicriviroc (CCR2/CCRSantagonist); Emricasan (Caspase inhibitors); GS-4997 (ASK1 inhibitor);Amlexanox (IKKε/TBK1 inhibitor); PXS-4728A (VAP-1 inhibitor); Orlistat(Intestinal lipase inhibitor); IMM-124e (IgG-rich bovine colostrum);Solithromycin (Antibiotic); Faecal microbial transplant (Modulation ofgut microbiome); Simtuzumab (LOXL2 antibody); GR-MD-02 (Galectin-3inhibitor); Trevogrumab (myostatin inhibitor); Garetosmab (activin Ainhibitor); and SRK-015 (myostatin inhibitor).

Such combination therapies may advantageously utilize lower dosages ofthe administered therapeutic agents, thus avoiding possible toxicitiesor complications associated with the various monotherapies.

Assays for Detecting a LTBP1-TGFβ1 Complex and/or a LTBP3-TGFβ1 Complex

In some embodiments, methods and compositions provided herein relate toa method for detecting a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1complex in a sample obtained from a subject. As used herein, a “subject”refers to an individual organism, for example, an individual mammal. Insome embodiments, the subject is a human In some embodiments, thesubject is a non-human mammal In some embodiments, the subject is anon-human primate. In some embodiments, the subject is a rodent. In someembodiments, the subject is a sheep, a goat, a cattle, poultry, a cat,or a dog. In some embodiments, the subject is a vertebrate, anamphibian, a reptile, a fish, an insect, a fly, or a nematode. In someembodiments, the subject is a research animal In some embodiments, thesubject is genetically engineered, e.g., a genetically engineerednon-human subject. The subject may be of either sex and at any stage ofdevelopment. In some embodiments, the subject is a patient or a healthyvolunteer.

In some embodiments, a method for detecting a LTBP1-TGFβ1 complex and/ora LTBP3-TGFβ1 complex in a sample obtained from a subject involves (a)contacting the sample with an antibody that selectively binds aLTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex under conditionssuitable for binding of the antibody to the antigen, if the antigen ispresent in the sample, thereby forming binding complexes; and (b)determining the level of the antibody bound to the antigen (e.g.,determining the level of the binding complexes).

In one embodiment, a screening assay that utilizes biotinylated latentTGFβ1 complexes immobilized onto a surface is utilized, which allows forthe activation of latent TGFβ by integrins, e.g., by providing a tether.Other, non-integrin activators could also be tested in that system. Areadout can be measured through reporter cells or other TGFβ-dependentcellular responses.

Cell-based assays for measuring TGFβ activation

Activation of TGFβ (and inhibition thereof by a TGFβ test inhibitor,such as an antibody) may be measured by any suitable method known in theart. For example, integrin-mediated activation of TGFβ can be utilizedin a cell-based assay, such as the “CAGA12” luciferase assay, describedin more detail herein. Such an assay system may comprise the followingcomponents: i) a source of TGFβ (recombinant, endogenous ortransfected); ii) a source of integrin (recombinant, endogenous, ortransfected); and iii) a reporter system that responds to TGFβactivation, such as cells expressing TGFβ receptors capable ofresponding to TGFβ and translating the signal into a readable output(e.g., luciferase activity in CAGA12 cells or other reporter celllines). In some embodiments, the reporter cell line comprises a reportergene (e.g., a luciferase gene) under the control of a TGFβ-responsivepromoter (e.g., a PAI-1 promoter). In some embodiments, certain promoterelements that confer sensitivity may be incorporated into the reportersystem. In some embodiments, such promoter element is the CAGA12element. Reporter cell lines that may be used in the assay have beendescribed, for example, in Abe et al. (1994) Anal Biochem. 216(2):276-84, incorporated herein by reference. In some embodiments, each ofthe aforementioned assay components are provided from the same source(e.g., the same cell). In some embodiments, two of the aforementionedassay components are provided from the same source, and a third assaycomponent is provided from a different source. In some embodiments, allthree assay components are provided from different sources. For example,in some embodiments, the integrin and the latent TGFβ complex (proTGFβand a presenting molecule) are provided for the assay from the samesource (e.g., the same transfected cell line). In some embodiments, theintegrin and the TGF are provided for the assay from separate sources(e.g., two different cell lines, a combination of purified integrin anda transfected cell). When cells are used as the source of one or more ofthe assay components, such components of the assay may be endogenous tothe cell, stably expressed in the cell, transiently transfected, or anycombination thereof.

A skilled artisan could readily adapt such assays to various suitableconfigurations. For instance, a variety of sources of TGFβ3 may beconsidered. In some embodiments, the source of TGFβ3 is a cell thatexpresses and deposits TGFβ3 (e.g., a primary cell, a propagated cell,an immortalized cell or cell line, etc.). In some embodiments, thesource of TGFβ3 is purified and/or recombinant TGFβ3 immobilized in theassay system using suitable means. In some embodiments, TGFβ3immobilized in the assay system is presented within an extracellularmatrix (ECM) composition on the assay plate, with or withoutde-cellularization, which mimics fibroblast-originated TGFβ3. In someembodiments, TGFβ3 is presented on the cell surface of a cell used inthe assay. Additionally, a presenting molecule of choice may be includedin the assay system to provide suitable latent-TGFβ complex. One ofordinary skill in the art can readily determine which presentingmolecule(s) may be present or expressed in certain cells or cell types.Using such assay systems, relative changes in TGFβ3 activation in thepresence or absence of a test agent (such as an antibody) may be readilymeasured to evaluate the effects of the test agent on TGFβ3 activationin vitro.

Such cell-based assays may be modified or tailored in a number of waysdepending on the TGFβ3 isoform being studied, the type of latent complex(e.g., presenting molecule), and the like. In some embodiments, a cellknown to express integrin capable of activating TGFβ3 may be used as thesource of integrin in the assay. Such cells include SW480/136 cells(e.g., clone 1E7). In some embodiments, integrin-expressing cells may beco-transfected with a plasmid encoding a presenting molecule of interest(such as GARP, LRRC33, LTBP (e.g., LTBP1 or LTBP3), etc.) and a plasmidencoding a pro-form of the TGFβ3 isoform of interest (such asproTGFβ31). After transfection, the cells are incubated for sufficienttime to allow for the expression of the transfected genes (e.g., about24 hours), cells are washed, and incubated with serial dilutions of atest agent (e.g., an antibody). In some embodiments, tissue culturewells or plates may be coated with a substance that provides a favorablesubstrate upon which cells may adhere, grow, and/or deposit ECMcomponents. This may facilitate ECM architecture, organization oradhesion of the cells thereto. For example, charged substances such aspoly-lysine may be used to pre-coad the tissue culture substrate.Additionally or alternatively, one or more components of ECM, such aslaminins, fibronectins, etc., may be used as substrate for coating. Insome embodiments, the cells are seeded on ECM protein-coatedwells/plates prior to transfection. In some embodiments, the cells areseeded on ECM coated wells/plates after transfection. In someembodiments, the wells/plates are coated with fibronectin. In someembodiments, the wells/plates are coated with human fibronectin.

After transfection (and optionally seeding on ECM coated well/plates), areporter cell line (e.g., CAGA12 cells) is added to the assay system,followed by appropriate incubation time to allow TGFβ signaling. Afteran incubation period (e.g., about 18-20 hours) following the addition ofthe test agent, signal/read-out (e.g., luciferase activity) is detectedusing suitable means (e.g., for luciferase-expressing reporter celllines, the Bright-Glo reagent (Promega) can be used). In someembodiments, Luciferase fluorescence may be detected using a BioTek(Synergy H1) plate reader, with autogain settings.

Kits for Use in Alleviating Diseases/Disorders Associated withLTBP1/3-TGFβ

The present disclosure also provides kits for use in alleviatingdiseases/disorders associated with a TGFβ-related indication. Such kitscan include one or more containers comprising an inhibitor, e.g.,antibody, or antigen-binding portion thereof, that selectively binds toa LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, e.g., any of thosedescribed herein.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise a description of administration of theinhibitor, e.g., antibody, or antigen-binding portion thereof, thatselectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex totreat, delay the onset, or alleviate a target disease as those describedherein. The kit may further comprise a description of selecting anindividual suitable for treatment based on identifying whether thatindividual has the target disease. In still other embodiments, theinstructions comprise a description of administering an antibody, orantigen-binding portion thereof, to an individual at risk of the targetdisease.

The instructions relating to the use of inhibitors, e.g., antibodies, orantigen-binding portions thereof, that selectively bind a LTBP1-TGFβ1complex and/or a LTBP3-TGFβ1 complex generally include information as todosage, dosing schedule, and route of administration for the intendedtreatment. The containers may be unit doses, bulk packages (e.g.,multi-dose packages) or sub-unit doses. Instructions supplied in thekits of the disclosure are typically written instructions on a label orpackage insert (e.g., a paper sheet included in the kit), butmachine-readable instructions (e.g., instructions carried on a magneticor optical storage disk) are also acceptable. The label or packageinsert can indicate that the composition is used for treating, delayingthe onset and/or alleviating a disease or disorder associated with aTGFβ-related indication. Instructions may be provided for practicing anyof the methods described herein.

The kits of this disclosure can be provided in suitable packaging.Suitable packaging includes, but is not limited to, vials, bottles,jars, flexible packaging (e.g., sealed Mylar or plastic bags), and thelike. Also contemplated are packages for use in combination with aspecific device, such as an inhaler, nasal administration device (e.g.,an atomizer) or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an inhibitor, e.g., antibody, orantigen-binding portion thereof, that selectively binds a LTBP1-TGFβ1complex and/or a LTBP3-TGFβ1 complex, as described herein.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the disclosure provides articles of manufacture comprisingcontents of the kits described above.

Diagnostics, patient selection, monitoring

Therapeutic methods that include TGFβ1 inhibition therapy may comprisediagnosis of a TGFβ1 indication and/or selection of patients likely torespond to such therapy. Additionally, patients who receive the TGFβ1inhibitor may be monitored for therapeutic effects of the treatment,which typically involves measuring one or more suitable parameters whichare indicative of the condition and which can be measured (e.g.,assayed) before and after the treatment and evaluating treatment-relatedchanges in the parameters. For example, such parameters may includelevels of biomarkers present in biological samples collected from thepatients. Biomarkers may be RNA-based, protein-based, cell-based and/ortissue-based. For example, genes that are overexpressed in certaindisease conditions may serve as the biomarkers to diagnose and/ormonitor the disease or response to the therapy. Cell-surface proteins ofdisease-associated cell populations may serve as biomarkers. Suchmethods may include the direct measurements of disease parametersindicative of the extent of the particular disease. Any suitablesampling methods may be employed, such as serum/blood samples, biopsies,and imaging.

While biopsies have traditionally been the standard for diagnosing andmonitoring various diseases, such as fibrosis (e.g., organ fibrosis) andproliferative disorders (e.g., cancer), less invasive alternatives maybe preferred. For example, many non-invasive in vivo imaging techniquesmay be used to diagnose, monitor, and select patients for treatment.Thus, the invention includes the use of in vivo imaging techniques todiagnose and/or monitor disease in a patient or subject. In someembodiments, the patient or subject is receiving an isoform-specificTGFβ1 inhibitor as described herein. In some embodiments, the patient orsubject is receiving an isoform-specific TGFβ1 inhibitor as describedherein. In other embodiments, an in vivo imaging technique may be usedto select patients for treatment with an isoform-specific TGFβ1inhibitor. In some embodiments, such techniques may be used to determineif or how patients respond to a therapy, e.g., TGFβ1 inhibition therapy.

Exemplary in vivo imaging techniques used for the methods include, butare not limited to X-ray radiography, magnetic resonance imaging (MRI),medical ultrasonography or ultrasound, endoscopy, elastography, tactileimaging, thermography, medical photography. Other imaging techniquesinclude nuclear medicine functional imaging, e.g., positron emissiontomography (PET) and Single-photon emission computed tomography (SPECT).Methods for conducting these techniques and analyzing the results areknown in the art.

Non-invasive imaging techniques commonly used to diagnose and monitorcancer include, but are not limited to: magnetic resonance imaging(MRI), computed tomography (CT), ultrasound, positron emissiontomography (PET), single-photon emission computed tomography (SPECT),fluorescence reflectance imaging (FRI), and fluorescence mediatedtomography (FMT). Hybrid imaging platforms may also be used to diagnoseand monitor cancer. For example, hybrid techniques include, but are notlimited to: PET-CT, FMT-CT, FMT-MRI, and PET-MRI. Dynamic contrastenhanced MRI (DCE-MRI) is another imaging technique commonly used todetect breast cancers. Methods for conducting these techniques andanalyzing the results are known in the art.

Non-invasive imaging techniques commonly used to diagnosis and monitorfibrosis include, but are not limited to: ultrasound (e.g., conventionalor contrast-enhanced ultrasound), ultrasound elastography (e.g.,transient elastography, point shear wave elastography and 2D-shear waveelastography), CT scan (e.g., conventional CT or CT perfusion imaging),magnetic resonance imaging (MRI) (e.g., conventional MRI, Magneticresonance elastography, diffusion weighted magnetic resonance imaging,gadoxetic acid disodium, and magnetic resonance perfusion imaging).

In some embodiments, non-invasive imaging techniques are used to assesslevels of liver fibrosis or hepatic steatosis. For example, imagingtechniques particularly useful to assess liver fibrosis may include butare not limited to: FibroScan (transient elastography; TE), point shearwave elastography (pSWE; a.k.a. acoustic radiation force impulse(ARFI)), 2D-3D SWE, magnetic resonance elastography (MRE), andmultiparameteric MRI. Imaging techniques particularly useful to assesshepatic steatosis may include but are not limited to: ultrasonography,controlled attenuation parameter (CAP) elastography, MRI-estimatedproton density fat fraction (MRI-PDFF), and magnetic resonancespectroscopy (MRS). In some embodiments, the in vivo imaging techniqueis used to assess liver stiffness. In some embodiments, the in vivoimaging technique is used to detect and assess intrahepatic triglyceridelevels. In some embodiments, in vivo imaging technique is used to assessliver surface nodularity (LSN; a.k.a. “liver score”), liver stiffness,and/or liver segmental volume ratio (LSVR), which are all beneficial inthe staging of hepatic fibrosis and sub-staging cirrhosis. Methods forconducting these techniques and analyzing the results are known in theart.

More recently, non-invasive imaging methods are being developed whichwill allow the detection of cells of interest (e.g., cytotoxic T cells,macrophages, and cancer cells) in vivo. See for example,www.imaginab.com/technology/; Tavare et al. (2014) PNAS, 111(3):1108-1113; Tavare et al. (2015) J Nucl Med 56(8): 1258-1264; Rashidianet al. (2017) J Exp Med 214(8): 2243-2255; Beckford Vera et al. (2018)PLoS ONE 13(3): e0193832; and Tavare et al. (2015) Cancer Res 76(1):73-82, each of which is incorporated herein by reference. So-called“T-cell tracking” is aimed to detect and localize anti-tumor effectorT-cells in vivo. This may provide useful insights into understanding theimmunosuppressive phenotype of solid tumors. Tumors that arewell-infiltrated with cytotoxic T cells (“inflammed” or “hot” tumors)are likely to respond to cancer therapies such as checkpoint blockadetherapy (CBT). On the other hand, tumors with immunosuppressivephenotypes tend to have poor T-cell infiltration even when there is ananti-tumor immune response. These so-called “immune excluded” tumorslikely fail to respond to cancer therapies such as CBT. T-cell trackingtechniques may reveal these different phenotypes and provide informationto guide in therapeutic approach that would likely benefit the patients.For example, patients with an “immune excluded” tumor are likely benefitfrom a TGFβ1 inhibitor therapy to help reverse the immunosuppressivephenotype. It is contemplated that similar techniques may be used todiagnose and monitor other diseases, for example, fibrosis. Typically,antibodies or antibody-like molecules engineered with a detection moiety(e.g., radiolabel, fluorescence, etc.) can be infused into a patient,which then will distribute and localize to sites of the particularmarker (for instance CD8+and M2 macrophages).

Non-invasive in vivo imaging techniques may be applied in a variety ofsuitable methods for purposes of diagnosing patients; selecting oridentifying patients who are likely to benefit from TGFβ1 inhibitortherapy; and/or, monitoring patients for therapeutic response upontreatment. Any cells with a known cell-surface marker may bedetected/localized by virtue of employing an antibody or similarmolecules that specifically bind to the cell marker. Typically, cells tobe detected by the use of such techniques are immune cells, such ascytotoxic T lymphocytes, regulatory T cells, MDSCs, disease-associatedmacrophages, (M2 macrophages such as TAMs and FAMs), NK cells, dendriticcells, and neutrophils.

Non-limiting examples of suitable immune cell markers include monocytemarkers, macrophage markers (e.g., M1 and/or M2 macrophage markers), CTLmarkers, suppressive immune cell markers, MDSC markers (e.g., markersfor G- and/or M-MDSCs), including but are not limited to: CD8, CD3, CD4,CD11b, CD163, CD206, CD68, CD14, CD15, CD66, CD34, CD25, and CD47.

In some embodiments, the in vivo imaging technique measures hepaticsteatosis, hepatic triglycerides, immune cells (e.g., as describedbelow), and/or myofibroblasts. In some embodiments, the treatmentreduces triglycerides, steatosis, liver surface nodules, inflammation,and/or macrophages, in the diseased tissue. In some embodiments, theselected patient has an intrahepatic triglyceride content of >5.5% ofliver volume, optionally wherein the intrahepatic triglyceride contentis >10% of liver volume. In some embodiments, the treatment reducesintrahepatic triglyceride content to <5.5% of liver volume. In someembodiments, the treatment reduces MDSCs in the diseased tissue. In someembodiments, the treatment reduces macrophages in the diseased tissue.In some embodiments, the effective amount is from 0.1 mg/kg to 30 mg/kg,optionally 3 mg/kg to 30 mg/kg. In some embodiments, the method furthercomprises monitoring the subject for a therapeutic response as describedherein (e.g., reduced triglycerides, reduced steatosis, reduced liversurface nodules, reduced inflammation, reduced macrophages, and/orreduced liver score).

Process of Screening; Manufacture

The invention encompasses screening methods, production methods andmanufacture processes of antibodies or fragments thereof which bind toand dissociates at slow rates from a hLTBP1-proTGFβ1 complex and/or ahLTBP3-proTGFβ1 complex, and pharmaceutical compositions and relatedkits comprising the same.

Methods for making a pharmaceutical composition comprising the antibody(or an engineered construct comprising an antigen-binding fragmentthereof) require identification and selection of such antibodies withdesirable attributes. Here, the invention includes the recognition thatantibodies with low k_(OFF) values may provide the durability thatreflects the mechanism of action of these activation inhibitors, whichdo not rely on the ability to rapidly compete binding with endogenousreceptors, but rather, exert inhibitory effects by latching ontoinactive latent forms of TGFβ1 within the tissue. The ability to staybound to the latent antigen complex (corresponding to low dissociationrates) may achieve durable potency in vivo.

Accordingly, the invention provides a method for manufacturing apharmaceutical composition comprising a TGFβ1-selective activationinhibitor, wherein the method comprises the steps of: selecting anantibody or antigen-binding fragment thereof that specifically binds ahuman LLC with a low dissociation rate (e.g., <5×10 (1/s)), andproducing the antibody at large-scale.

The selection of inhibitors with favorable off rates (low dissociation)may be determined with monovalent antibodies (e.g., Fab fragments) orfull-length antibodies (e.g., IgGs).

In some embodiments, the step of producing comprises a mammalian cellculture having a volume of 250L or greater, e.g., 1000 L, 2000 L, 3000L, 4000 L. The method may further comprise the step of purifying theantibody from the cell culture, and optionally formulating the purifiedantibody into a pharmaceutical composition. In some embodiments, themethod further comprises the step of testing the selected antibody in asuitable preclinical model for efficacy and safety and confirming thatthe antibody is efficacious at a NOAEL. The safety assessment mayinclude in vivo toxicology study comprising histopathology andimmune-directed safety assessment including, for example, in vitrocytokine release assays and platelet assays.

In order to achieve durable inhibitory effects, antibodies withdissociation rates (e.g., monovalent dissociation rates) of no greaterthan 10.0E-4 (s⁻¹) (e.g., 5.0E-4 or less, 1.0E-4 or less, 5.0E-5 orless) may be selected for therapeutic use and/or large-scale manufacturein accordance with the present disclosure.

While several embodiments of the present disclosure have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the presentdisclosure. More generally, those skilled in the art will readilyappreciate that all parameters, dimensions, materials, andconfigurations described herein are meant to be exemplary and that theactual parameters, dimensions, materials, and/or configurations willdepend upon the specific application or applications for which theteachings of the present disclosure is/are used. Those skilled in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of thedisclosure described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, thedisclosure may be practiced otherwise than as specifically described andclaimed. The present disclosure is directed to each individual feature,system, article, material, and/or method described herein. In addition,any combination of two or more such features, systems, articles,materials, and/or methods, if such features, systems, articles,materials, and/or methods are not mutually inconsistent, is includedwithin the scope of the present disclosure.

The present invention is further illustrated by the following examples,which are not intended to be limiting in any way. The entire contents ofall references, patents and published patent applications citedthroughout this application, as well as the Figures, are herebyincorporated herein by reference.

EXAMPLES

Transforming growth factor beta 1 (TGFβ31) is expressed as a pro-proteinthat is proteolytically cleaved into a C-terminal growth factor and anN-terminal prodomain After cleavage, the prodomain remains noncovalentlyassociated with the growth factor, preventing receptor binding. Thislatent TGFβ1 forms a large latent complex (LLC) through disulfide bondsthat link the prodomain to presenting molecules, and these large latentcomplexes are then deposited into the extracellular matrix (ECM) orbrought to the cell surface. These presenting molecules provide ananchor for specific αVβ integrins to exert traction force on latentTGFβ1. Four TGFβ1 presenting proteins have been identified: Latent TGFβBinding Protein-1 (LTBP1) and LTBP3 are deposited in the extracellularmatrix, while Glycoprotein-A Repetitions Predominant (GARP/LRRC32) andLeucine-Rich Repeat-Containing Protein 33 (LRRC33) present latent TGFβ1on the surface of immune cells. TGFβ1 is involved in tissue homeostasisprocesses and regulation of immune responses, and dysregulation of itsactivation is a key driver of organ fibrosis, cancer, and autoimmunity.

As compared to the TGFβ growth factors and the receptors, which areexpressed broadly, the four presenting molecule-proTGFβ complexes,namely, LTBP1-proTGFβ, LTBP3-proTGFβ, GARP-proTGFβ and LRRC33-proTGFβ,show more restricted or selective (e.g., tissue-specific) expressionpatterns, giving rise to functional compartmentalization of TGFβactivities by virtue of association. The presenting molecule-proTGFβcomplexes therefore provide discrete “contexts” of TGFβ signaling withinthe tissue in which the presenting molecules are expressed. Thesecontexts may be divided into two broad categories: i) TGFβ signalingassociated with the ECM (e.g., matrix-associated TGFβ function); and ii)TGFβ signaling associated with cells (particularly certain immune cellfunction). The LTBP1-proTGFβ and LTBP3-proTGFβ complexes fall under thefirst category, while GARP-proTGFβ and LRRC33-proTGFβ complexes fallunder the second category.

Non-selective targeting of TGFβ activity for therapeutic purposes hasbeen challenging due to dose-limiting toxicities reported for pan-TGFβpathway inhibitors, as well as immune system activation through chronicTGFβ suppression. In an effort to address this therapeutic need for bothisoform- and context-selectivity for TGFβ1 targeting, provided hereinare inhibitors of TGFβ that are capable of selectively inhibiting theactivation of TGFβ that is associated with the ECM. In some embodiments,the inhibitors are also selective for a particular TGFβ isoform (e.g.,proTGFβ1, proTGFβ2, and/or proTGFβ33). The isoform-specific monoclonalantibodies bind the latent TGFβ1 prodomain, with no detectable bindingto latent TGFβ2 or TGFβ3, and inhibit integrin-mediated activation oflatent TGFβ1 in vitro with context-selectivity. In order to facilitateantibody discovery and characterization efforts, context-dependentcell-based assays of TGFβ1 activation were developed.

Example 1 Development of Context-Specific Inhibitors that Bind aLTBP1/3-TGFβ1 Complex

SR-AB1 was used as a control. SR-AB1 binds latent TGFβ1 independent ofthe presenting molecule (see FIG. 2A).

Antibodies that are selective for TGFβ1-containing large latentcomplexes were developed. SR-AB2 was selected for further analysis usingthe functional assays described in the below examples. The heavy andlight chain variable regions of SR-AB2 were sequenced (FIG. 8);complementarity determining regions are underlined. It was demonstratedthat SR-AB2 binds LTBP-presented latent TGFβ1 complexes but does notbind GARP-TGFβ1 or proTGFβ1 alone (FIG. 2B). However, as describedbelow, the functional effect of such selective binding was unknown andcould not be determined using currently known techniques without thefurther development of novel functional assays.

Example 2 Functional Assays to Detect Inhibition of ActivatedRecombinant Latent TGFβ1

In order to identify isoform-specific inhibitors that bind the latentTGFβ1 prodomain with no detectable binding to latent TGFβ2 or TGFβ3 andthat inhibit integrin-mediated activation of latent TGFβ1 in vitro withcontext-dependency, new functional assays were required. Prior to theinstant invention, assays were not available which could detectisoform-specific TGFβ1 antibodies that bound only to LTBPs.Specifically, previous assay formats could not differentiate between theactivation of proTGFβ1 presented by endogenous presenting molecules andthe activation of proTGFβ1 presented by exogenous LTBPs. By directlytransfecting integrin-expressing cells, the novel assays disclosedherein establish a window between endogenous presenter-proTGFβ1 activityand exogenous LTBP-proTGFβ1 activity. As LTBP-proTGFβ1 complexes areembedded in the extracellular matrix, the assay plate coating is also animportant component of the assay. The use of high binding plates, coatedwith the ECM protein Fibronectin, made the LTBP assays more robust. Inother words, prior to the instant disclosure, there was no assay windowbetween proTGFβ1 transfection and co-transfection of LTBP1/3 +proTGFβ1.Prior to the instant invention, the only available assay format was atriple co-culture system: transfectants (latent TGFβ presenting cells)+integrin expressing cells (activator) +CAGA cells (reporter). Bycombining the first two cell populations, and directly transfecting theintegrin expressing cells with TGFβ and presenting molecules, a windowfor LTBP-proTGFβ1 activation was established herein.

The issue of ‘bulk transfection’ (i.e., transfection in a separatewell/plate/dish prior to seeding in the assay well) vs ‘directtransfection’ (i.e., transfection in the assay well) protocol andwhether an assay window is seen for LTBP complexes seems to be celldependent in some situations. Thus, the discovery and characterizationof LTBP1/3-TGFβ1 inhibitors, e.g., antibodies and antigen-bindingportions thereof, would not have been possible without the developmentof context-dependent cell-based assays of TGFβ1 activation describedherein (see also FIG. 3A and 3B).

Specifically, to determine if the antibodies developed in Example 1 werefunctional, cell-based assays of αVβ integrin activation of TGFβ1 largelatent complex (LLC) were developed, which are specific for each knownpresenting molecule: LTBP1, LTBP3, GARP and LRRC33. Through the processof assay development and optimization, it was determined thatfibronectin is a critical ECM protein for the integrin-dependent invitro activation of LTBP presented TGFβ1 LLCs. The context-independentand LTBP complex-specific TGFβ1 LLC antibodies were also validated asinhibitors of integrin-dependent activation using the below assays.Thus, the antibodies developed in Example 1 can be divided into 2classes: antibodies which bind all TGFβ1 containing complexes(isoform-specific and context independent), and antibodies which onlybind LTBP presented TGFβ1 LLC. As described in more detail herein, thedevelopment of an LTBP complex-specific class of inhibitor, which wasnot capable of being identified prior to the assays developed anddescribed herein, enables a therapeutic approach for treating fibroticindications, and could allow for chronic dosing while avoiding immunesystem activation due to TGFβ1 inhibition of immune suppressive cells.

Assay I. Activation of Latent TGF/β1 using SW4801/β Cells

For the assay depicted in FIG. 3A, the following protocol was developed.This assay is optimal for extracellular matrix (LTBP presented)activation by integrin cells.

Materials:

MvLul-CAGA12 cells (Clone 4A4)

SW480/136 cells (Clone 1E7) (aV subunit is endogenously expressed athigh levels; 136 subunit is stably overexpressed)

Costar white walled TC treated 96 well assay plate #3903

Greiner Bio-One High Binding white μclear 96 well assay plate #655094

Human Fibronectin (Corning #354008)

P200 multichannel pipet

P20, P200, and P1000 pipets with sterile filter tips for each

Sterile microfuge tubes and rack

Sterile reagent reservoirs

0.4% trypan blue

2 mL, 5 mL, 10 mL, and 25 mL sterile pipets

Tissue culture treated 100 mm or 150 mm plates

70%Ethanol

Opti-MEM reduced serum media (Life Tech #31985-070)

Lipofectamine 3000 (Life Tech #L3000015)

Bright-Glo luciferase assay reagent (Promega #E2620)

0.25% Trypsin +0.53mM EDTA

proTGFb1 expression plasmid, human (SR005)

LTBP1S expression plasmid, human (SR044)

LTBP3 expression plasmid, human (SR117)

LRRC32 (GARP) expression plasmid, human (SR116)

LRRC33 expression plasmid, human (SR386)

Equipment:

BioTek Synergy H1 plate reader

TC hood

Bench top centrifuge

CO₂ incubator 37° C. 5% CO₂

37° C. water/bead bath

Platform shaker

Microscope

Hemocytometer/countess

Definitions:

CAGA12 4A4 cells: Derivative of MvLul cells (Mink Lung EpithelialCells), stably transfected with CAGA12 synthetic promoter, drivingluciferase gene expression

DMEM-0.1%BSA: Assay media; base media is DMEM (Gibco Cat# 11995-065),media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin,and 4mM glutamine

D10: DMEM 10% FBS, P/S, 4mM glutamine, 1% NEAA, 1X GlutaMAX (Gibco Cat#35050061)

SW480/β6 Media: D10 +1000 μg/mL G-418

CAGA12 (4A4) media: D10 +0.75 μg/mL puromycin

Procedure:

On Day 0, cells were seeded for transfection. SW480/β6 (clone 1E7) cellswere detached with trypsin and pelleted (spin 5 min @ 200×g). Cellpellet was resuspended in D10 media and viable cells per ml werecounted. Cells were seeded at 5.0e6 cells/12m1/100 mm TC dish. ForCAGA12 cells, cells were passaged at a density of 1 0 million per T75flask, to be used for the assay on Day 3. Cultures were incubated at 37°C. and 5% CO_(2.)

On Day 1, integrin-expressing cells were transfected. Manufacturer'sprotocol for transfection with Lipofectamine 3000 reagent was followed.Briefly, the following were diluted into OptiMEM I, for 125 μl per well:7.5 μg DNA (presenting molecule) +7.5 μg DNA (proTGFβ31), 30 μl P3000,and up to 125 μl with OptiMEM I. The well was mixed by pipetting DNAtogether, then OptiMEM was added. P3000 was added, and everything wasmixed well by pipetting. A master mix of Lipofectamine3000 was made, tobe added to DNA mixes: for the LTBP1 assay: 15 μl Lipofectamine3000, upto 125 μl in OptiMEM I, per well; for the LTBP3 assay: 45 μlLipofectamine3000, up to 125 μl in OptiMEM I, per well. DilutedLipofectamine3000 was added to DNA, mixed well by pipetting, andincubated at room temp for 15 min. After the incubation, the solutionwas mixed a few times by pipetting, and then 250 μl ofDNA:Lipofectamine3000 (2×125 μl) per dish was added dropwise. Each dishwas gently swirled to mix and the dish was returned to the tissueculture incubator for—24hrs.

On Days 1-2, the assay plates were coated with human fibronectin.Specifically, lyophilized fibronectin was diluted to lmg/ml inultra-pure distilled water (sterile). lmg/ml stock solution was dilutedto 19.2 μg/ml in PBS (sterile). 50 μl/well was then added to the assayplate (high binding) and incubated 0/N in tissue culture incubator (37°C. and 5% CO₂). Final concentration was 3.0 μg/cm².

On Day 2, transfected cells were plated for assay and inhibitoraddition. First, the fibronectin coating was washed by adding 200 μlwell PBS to the fibronectin solution already in the assay plate. Removedwash manually with multichannel pipette. Wash was repeated for twowashes total. The plate was allowed to dry at room temperature with lidoff prior to cell addition. The cells were then plated by detaching withtrypsin and pelleted (spin 5 min @ 200×g.). The pellet was resuspendedin assay media and viable cells were counted per ml. For the LTBP1 assaycells were diluted to 0.10e6cells/ml and seeded 50 μl per well (5,000cells per well). For the LTBP3 assay, cells were diluted to 0.05e6cells/ml and seeded 50 μl per well (2,500 cells per well). To preparefunctional antibody dilutions, antibodies were pre-diluted to aconsistent working concentration in vehicle. Stock antibodies wereserially diluted in vehicle (PBS is optimal, avoid sodium citratebuffer). Each point of serial dilution was diluted into assay media fora 4X final concentration of antibody. 25 μl of 4X antibody was added perwell and cultures were incubated at 37° C. and 5% CO₂ for —24 hours.

On Day 3, the TGFβ3 reporter cells were added. CAGA12 (clone 4A4) cellsfor the assay were detached with trypsin and pelleted (spin 5 min @200×g.). The pellet was resuspended in assay media and viable cells perml were counted. Cells were diluted to 0.4e⁶cells/ml and seeded 50 μlper well (20,000 cells per well). Cells were returned to incubator.

On Day 4, the assay was read (16-20 hours after antibody and/or reportercell addition). Bright-Glo reagent and test plate were allowed to cometo room temperature before reading. Read settings on BioTek Synergy H1were set using TMLC_std protocol—this method has an auto-gain setting.Selected positive control wells for autoscale (high). 100 μL ofBright-Glo reagent was added per well. Incubated for 2 min with shaking,at room temperature, protected plate from light. The plate was read onBioTek Synergy H1.

In some embodiments, TGFβ3 activity associated with endogenouspresenting molecules (e.g., LTBP1/3) may be assessed by onlytransfecting proTGFβ1 (i.e., without co-transfecting LTBP1/3). In someembodiments, the presenting molecule and proTGFβ3 DNA may be directlytransfected into SW480/136 cells seeded in an assay well (i.e., “directtransfection”), rather than transfecting the cells in a separatewell/dish/plate as essentially described above (i.e., “bulktransfection”). See also Assay II below for direct transfectionprotocol. In some embodiments, SW480/136 cells may be seeded in assaywells without fibronectin as essentially described in Assay II below.

Results:

Data generated from this assay reflected TGFβ3 activity in cellsupernatants (FIG. 3A). Specifically, SW480/136 cells were bulktransfected with LTBP1/3 and proTGFβ1, and seeded on fibronectin asdescribed above. Raw data units were relative light units (RLU). FIG. 3Ademonstrates that transfection of LTBP1-proTGFβ1 and/or LTBP3-proTGFβ1,but not proTGFβ1 alone, induces a TGFβ3 activation signal.

The assay was further optimized as described in FIG. 4. Specifically,the relative contribution of presenting molecule and/or proTGFβ1 tolatent TGFβ1 activation was determined. In this assay, SW480/β6 cellswere bulk transfected with the indicated DNA molecules and seeded onpre-coated assay wells with fibronectin as essentially described above.As shown in FIG. 4A, a significant increase in latent TGFβ1 activationupon co-transfection of presenting molecule and proTGFβ1 was observed.FIG. 4B depicts the optimization of co-transfection by changing theratio of plasmid DNAs for presenting molecule and proTGFβ1. Equivalentamounts of each plasmid were found to be optimal for co-transfection.

FIG. 5 demonstrates that fibronectin promotes integrin activation ofLTBP-presented latent TGFβ1. In this assay, SW480/β36 cells were bulktransfected with the indicated DNA molecules and seeded on pre-coatedassay well with varying concentrations of fibronectin purified fromhuman plasma. Fibronectin increased activation of latent TGFβ presentedby LTBP1 and LTBP3.

Assay II. Activation of Latent TGFβ1 using LN229 cells

For the assay depicted in FIG. 3B, the following protocol was developed.This assay, or “direct-transfection” protocol, is optimal forcell-surface presented TGFβ1 (GARP or LRRC33 presenter) activation byintegrin cells. LN229 cells express integrin αVβ8 (as opposed to theSW480b6 cell line, which was engineered to express αVβ6.

These two cell lines enable testing of antibodies on latent TGFβ1activated by either of the two best validated TGFβ-activating integrins.

Materials:

MvLu1-CAGA12 cells (Clone 4A4)

LN229 cell line (high levels of endogenous αVβ8 integrin)

Costar white walled TC treated 96 well assay plate #3903

Greiner Bio-One High Binding white μclear 96 well assay plate #655094

Human Fibronectin (Corning #354008)

P200 multichannel pipet

P20, P200, and P1000 pipets with sterile filter tips for each

Sterile microfuge tubes and rack

Sterile reagent reservoirs

0.4% trypan blue

2 mL, 5 mL, 10 mL, and 25 mL sterile pipets

Tissue culture treated 100 mm or 150 mm plates

70%Ethanol

Opti-MEM reduced serum media (Life Tech #31985-070)

Lipofectamine 3000 (Life Tech #L3000015)

Bright-Glo luciferase assay reagent (Promega #E2620)

0.25% Trypsin +0.53mM EDTA

proTGFb1 expression plasmid, human (SR005)

LTBP1S expression plasmid, human (SR044)

LTBP3 expression plasmid, human (SR117)

LRRC32 (GARP) expression plasmid, human (SR116)

LRRC33 expression plasmid, human (SR386)

Equipment:

BioTek Synergy H1 plate reader

TC hood

Bench top centrifuge

CO₂ incubator 37° C. 5% CO2

37° C. water/bead bath

Platform shaker

Microscope

Hemocytometer/countess

Definitions:

CAGA12 4A4 cells: Derivative of MvLul cells (Mink Lung EpithelialCells), stably transfected with CAGA12 synthetic promoter, drivingluciferase gene expression

DMEM-0.1%BSA: Assay media; base media is DMEM (Gibco Cat# 11995-065),media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin,and 4mM glutamine

D10: DMEM 10% FBS, P/S, 4mM glutamine, 1% NEAA, 1X GlutaMAX (Gibco Cat#35050061)

CAGA12 (4A4) media: D10 +0.75ug/mL puromycin

Procedure:

On Day 0, integrin expressing cells were seeded for transfection. Cellswere detached with trypsin and pelleted (spin 5 min @ 200×g). Cellpellet was resuspended in D10 media and count viable cells per ml. Cellswere diluted to 0.1e⁶ cells/ml and seeded 100 μl per well (10,000 cellsper well) in an assay plate. For CAGA12 cells, passaged at a density of1 5 million per T75 flask, to be used for the assay on Day 2. Cultureswere incubated at 37° C. and 5% CO_(2.)

On Day 1, cells were transfected. The manufacturer's protocol wasfollowed for transfection with Lipofectamine 3000 reagent. Briefly, thefollowing was diluted into OptiMEM I, for 5 μl per well: 0.1 μg DNA(presenting molecule) +0.1 μg DNA (proTGFβ31), 0.4 μl P3000, and up to 5μl with OptiMEM I. The well was mixed by pipetting DNA together, thenadd OptiMEM. Add P3000 and mix everything well by pipetting. A mastermix was made with Lipofectamine3000, to be added to DNA mixes: 0.2 μlLipofectamine3000, up to 5 μl in OptiMEM I, per well. DilutedLipofectamine3000 was added to DNA, mixed well by pipetting, andincubated at room temp for 15 min. After the incubation, the solutionwas mixed a few times by pipetting, and then 10 μl per well ofDNA:Lipofectamine3000 (2×5 μl) was added. The cell plate was returned tothe tissue culture incubator for—24hrs.

On Day 2, the antibody and TGFβ3 reporter cells were added. In order toprepare functional antibody dilutions, stock antibody in vehicle (PBS isoptimal) was serially diluted. Then each point was diluted into assaymedia for 2X final concentration of antibody. After preparingantibodies, the cell plate was washed twice with assay media, byaspirating (vacuum aspirator) followed by the addition of 100 μl perwell assay media. After second wash, the assay media was replaced with50 μl per well of 2X antibody. The cell plate was returned to theincubator for—15-20 min.

In order to prepare the CAGA12 (clone 4A4) cells for the assay, thecells were detached with trypsin and pelleted (spin 5 min @ 200×g.). Thepellet was resuspended in assay media and viable cells per ml werecounted. Cells were diluted to 0.3e⁶cells/ml and seeded 50 μl per well(15,000 cells per well). Cells were returned to incubator.

On Day 3, the assay was read about 16-20 hours after the antibody and/orreporter cell addition. Bright-Glo reagent and test plate were allowedto come to room temperature before reading. The read settings on BioTekSynergy H1 were set to use TMLC_std protocol—this method has anauto-gain setting. Positive control wells were set for autoscale (high).100 μL of Bright-Glo reagent was added per well. Incubated for 2 minwith shaking, at room temperature, protected plate from light. The platewas read on BioTek Synergy H1.

In some embodiments, TGFβ3 activity associated with endogenouspresenting molecules (e.g., LTBP1/3) may be assessed by onlytransfecting proTGFβ1 (i.e., without co-transfecting LTBP1/3). In someembodiments, the presenting molecule and proTGFβ3 DNA may be bulktransfected into LN229 cells in a separate well/dish/plate (i.e., “bulktransfection”) as essentially described above in Assay I, rather thandirectly transfecting the cells (i.e., “direct transfection”). In someembodiments, LN229 cells may be seeded in assay wells with fibronectin(see Assay I for fibronectin pre-coating protocol).

Data generated from this assay reflects TGFβ1 activity in cellsupernatants (FIG. 3B). Specifically, LN229 cells were seeded in assaywells without fibronectin and transfected with the indicated DNAmolecules by “direct transfection”. Raw data units are relative lightunits (RLU). Samples with high RLU values contained high amounts of freeTGFβ3, samples with low RLU values contained low levels of TGFβ3.

Example 3 SR-AB1 Is A Context-Independent Inhibitor of TGF/3 LLCActivation by Integrin

FIG. 6 is a graph demonstrating that SR-AB1 is a context-independentinhibitor of TGFβ1 large latent complex (LLC) by integrin. In thisassay, SW480/136 cells were seeded in assay wells without fibronectinand directly transfected with GARP-proTGFβ1 or LRRC33-proTGFβ1,respectively, as essentially described in the above protocols in Example2. To assess activation of TGFβ1 by LTBP1, LN229 cells were seeded inassay wells pre-coated with fibronectin and directly transfected withLTBP1-proTGFβ1, as essentially described in the above protocols inExample 2. SR-AB1 was shown to inhibit integrin activation of TGFβindependent of the presenting molecule.

Example 4 SR-AB2 Is A Complex-Specific Inhibitor of LTBP-proTGFβ1

SR-AB2 was selected for further analysis and testing for specificity forbinding to different TGFβ presenting molecules. Initially, an ELISAassay was conducted to test complex-specificity as follows.

Materials:

Solid white 96-well plates from the NeutrAvidin Coating of 96-WellPlates SOP

Biotinylated antigen

Maine Biotechnology Services Anti-His antibody MAB230P

Jackson ImmunoResearch Laboratories Peroxidase Affinipure Goat a-humanFCγ

Fragment Specific. Catalogue number 109-035-008.

Jackson ImmunoResearch Laboratories Affinipure Goat a-mouse FCγ FragmentSpecific.

Catalogue number 115-035-008

QuantaBlu ELISA Substrate (Pierce Biotech catalog number 15162)

Equipment:

Multi-channel pipette

P200 tips

Tabletop ultracentrifuge

1.5 mL centrifuge tubes

0.5 mL centrifuge tubes

P1000

P1000 tips

P200

P10

P10 tips

15 mL Falcon tubes

50 mL Falcon tubes

Biotek ELx 405 Select CW plate washer

Multidrop

Biotek Synergy H1 plate reader

Definitions:

Wash buffer: TBS (Tris-Buffered Saline; 50 mM Tris-Cl, 150 mM NaCl, pH7.6) with 0.05% Tween-20. For manual (hand) wash add 0.1% BSA as BSA issticky and should not be used with the automated plate washer system.

Sample Buffer: TBS (Tris-Buffered Saline; 50 mM Tris-Cl, 150 mM NaCl, pH7.6) with 0.05% Tween-20 and 0.1% BSA.

ELISA 3X protocol: Wash protocol on the Biotek ELx 405 Select CW platewasher. Washes with 2004, of wash buffer. Repeats the wash twoadditional times. Specifications: 3 cycles. No shaking. Dispenses 200 μLper well. Dispense flow rate setting 7 (range 1-10). Dispense height15.24 mm Horizontal x dispense position 0 mm Horizontal y dispenseposition 0 mm Aspirate height 3.048 mm horizontal x aspirate position1.372 mm horizontal aspirate y position 0.452 mm Aspiration rate 3.4mm/second. Aspiration delay 0 milliseconds. Crosswire aspiration onfinal wash. Crosswire height 3.048 mm Crosswire horizontal x position:-1.829 mm Crosswire horizontal y position: -0.457 mm

Procedure:

Remove a pre-coated and pre-blocked 96-well plate from 4° C. Dump outthe 1xPBS pH 7.4 1%BSA with 0.1% Tween-20 from the plate and forcefullyhit the plate on a Styrofoam pad lined with paper towels. If a 96-wellplate is not already prepared, prepare one with the NeutrAvidin Coatingof 96-Well Plates protocol. Specifically, for one NeutrAvidin coatedplate, remove 5 μL from the top of the 1 mg/mL NeutrAvidin stocksolution and dilute it into 10 mL of 1× carbonate buffer pH 9.4. Mix byinverting the falcon tube. Using a multi-channel pipette, place 100 μLin each well of the Corning high binding 96-well assay plate andincubate the 96-well plate overnight at 4° C. Wash the 96-well platewith 200 μL of wash buffer per well and repeat this step two additionaltimes. The plate should be washed for a total of three times. Block theplate with 200 μL per well of 1% BSA in PBS pH 7.4 and incubate theplate for 1 hour at 37° C. or overnight at 4° C.

Dilute biotinylated antigen in sample buffer. Optimal captureconcentration should first be determined by titration for eachindividual protein. Add 50 μl per well and incubate at room temperaturefor 1 hour.

Wash plates using the plate washer with wash buffer using the ELISA 3Xprotocol.

Dilute antibody in sample buffer. Screening of antibodies is performedat 1 μg/mL. Prepare α-His coating control antibody (Maine BiotechnologyServices catalogue number: MAB230P) at 1 μg/mL in sample buffer. Place50 μL of diluted antibody on designated wells and incubate at roomtemperature for 1 hour.

Wash plates using the plate washer with wash buffer using the ELISA 3Xprotocol.

Dilute human secondary antibody (Jackson ImmunoResearch LaboratoriesPeroxidase

Affinipure Goat a-human FCy Fragment Specific. Catalogue number109-035-008) 1:10,000 in sample buffer. For a-his wells, dilute mousesecondary antibody (Jackson ImmunoResearch Laboratories Affinipure Goata-mouse FCy Fragment Specific. Catalogue number 115-035-008) 1:10,000 insample buffer.

Place 50 μL of diluted antibody on designated wells. Incubate at roomtemperature for 1 hour. Wash plates using the plate washer with washbuffer using the ELISA 3X protocol.

Prepare SuperSignal ELISA Femto Substrate (Pierce Biotech catalog number15162) working solution according to manufacturer's protocol. 10 mL willbe needed for one plate. Place 100 μL QuantBlu Substrate workingsolution per well. Incubate for 10 minutes at room temperature.

Measure relative fluorescent units (RFU's) on a plate reader with anexcitation of 325nm and emission of 420nm.

Results:

FIG. 7A demonstrates that SR-AB2 only binds LTBP-proTGFβ1 complex; itdoes not bind proTGFβ1 or LTBP1 alone by ELISA. FIG. 7A demonstratesthat SR-AB2 does not bind GARP-proTGFβ1 by ELISA.

Additionally, the ability of SR-AB2 to inhibit LTBP1/3-proTGFβ1 in acell-based assay was also conducted. As essentially described above inExample 2, LN229 cells were seeded on fibronectin pre-coated assay wellsand directly transfected with the indicated DNA molecules. FIG. 7Bdepicts that SR-AB2 inhibits integrin activation of LTBP1-proTGFβ1(human and mouse complexes). FIG. 7C depicts that SR-AB2 inhibitsintegrin activation of LTBP3-proTGFβ1.

It was demonstrated that SR-AB2 specifically binds to proTGFβ1:LTBP1 & 3complexes, and not GARP-TGFβ1 or GARP-Lap complexes by ELISA asessentially described above (see FIG. 9).

As discussed above, LTBP1 and LTBP3 are deposited in the extracellularmatrix, while GARP/LRRC32 and LRRC33 present latent TGFβ1 on the surfaceof immune cells. It was demonstrated that SR-AB2 inhibits LTBP-proTGFβ1signaling, but does not affect GARP-proTGFβ1 (FIG. 10A and FIG. 10B).FIG. 10A demonstrates that SR-AB2 inhibits LTBP-proTGFβ presented byendogenous LTBP1/3. This assay was performed in LN229 cells, which wereseeded on fibronectin pre-coated wells and directly transfected withproTGFβ1. FIG. 10B demonstrates that SR-AB2 does not inhibitGARP-proTGFβ. SR-AB1 binds latent TGFβ1 independent of the presentingmolecule. This assay was performed in LN229 cells, were seeded in assaywells without fibronectin and directly transfected with GARP-proTGFβ1.

Inhibition of LTBP-proTGFβ1 by SR-AB2 was also shown in αVβ6Integrin-dependent activation of LTBP1-presented TGFβ1 in cell-basedassays (human and mouse, data not shown). SR-AB2 showed no inhibitoryeffects on overexpressed LRRC33-proTGFβ1.

Example 5 Octet binning of LTBP1-proTGF A1 antibodies

500 nM of human LTBP1-proTGFb1 complex was pre-incubated with 1 μM ofeach test antibody. After an overnight incubation, theLTBP1-proTGFβ1+first antibody was tested for binding to a secondantibody which was immobilized to an Anti-Human IgG Fc Capture (AHC)sensor tip at 67 nM. The sensor tip was blocked with a negative controlantibody (HuNeg) before seeing the LTBP1-proTGFβ1+first antibody.

Binding of the complex to a specific second antibody was normalized tothe uninhibited interaction, that is the complex in the presence of anegative control antibody (HuNeg) that does not bind to the TGFβcomplex. Normalized responses less than 70% or less than 0.7 of theuninhibited interaction were considered antibodies that cross block. Aresponse greater than 1 indicated that both antibodies were boundsimultaneously. The results are shown in Table 7, below.

TABLE 7 Second SR-AB13 SR-AB10 SR-AB2 SR-AB1 Antibody SR-AB13 0.47 1.381.29 1.08 SR-AB10 1.27 0.51 1.53 0.82 SR-AB2 1.31 1.54 0.47 1.11 SR-AB11.43 0.76 1.38 0.69 HuNeg 1 1 1 1 First Antibody

As shown in Table 7, antibodies SR-AB13, SR-AB10, SR-AB2 and SR-AB1 donot cross block each other, and therefore each antibody occupies adistinct epitope on the surface of human LTBP1-proTGFβ1.

Example 6 In vitro Binding Profile and Affinity Data

Suitable methods for in vitro binding assays to determine the parametersof binding kinetics include Bio-layer Interferometry (BLI)-based assayssuch as Octet, and surface plasmon resonance (SPR)-based assays, such asBiacore systems (see, for example: http://www.biophysics.bioc. cam.ac.uk/wp-content/uploads/2011/02/Biacore_assay_handbook.pdf).

The affinity of SR-AB10, SR-AB2 and SR-AB13 was measured by Octet assay.The protocol used to measure the affinity of the antibodies to thecomplexes provided herein is summarized below.

Materials:

96 well black polypropylene plates

AHC Octet tips (anti-human IgG Fc capture tips) (ForteBio)

10× kinetics buffer (ForteBio) (diluted to 1× in PBS)

reiner Bio-One 96-Well Half Area Microplates (VWR cat# 82050-044)

Procedure:

Notes: The volume within each well during an Octet experiment is 100 uL.A shake speed of 1000 rpm is used for all assay steps.

Pre-Wet Tips:

Biosensors must be pre-wet in lx kinetics buffer (1×KB) for at least 10minutes before starting an experiment. This can be done inside the Octetor on the bench.

Loading:

Tips are baselined in 1×KB for 1 minute before loading.

AHC tips are loaded with antibody at a concentration of 1 μg/mL (˜7 nM)for 3 minutes. A limit is set so that loading will stop when any onesensor reaches a response of 1 nanometer.

Tips are baselined in buffer for 1 minute after loading. The antibodyshould not dissociate from the tips during this time.

Antigen Association:

TGFβ1 in complex with various presentation molecules was associated tothe immobilized antibodies at a single concentration of 100 nM in 1×KB.

Antibody Dissociation:

Dissociation in 1×KB was performed for 3 minutes.

Data Analysis using ForteBio Data analysis software 8.2:

Processing: align Y axis to last 5 seconds of baseline, performinter-step correction with align to dissociation, and performSavitzky-Golay filtering.

Analysis: 1:1 fitting model is utilized. Fitting is local and full.(Local indicates each antibody is evaluated separately and fullindicates that both association and dissociation are considered) Fit thecurves and then save the report/export the data.

Results:

FIG. 11 presents the binding profile and affinity data for LTBPcomplex-specific antibodies SR-AB10, SR-AB2, and SR-AB13. Notably,SR-AB13 binds both human LTBP1 and human LTBP3 complexed with proTGFβ1,while SR-AB2 and SR-AB10 are specific to human LTBP1 complexed withTGFβ1.

Example 7 Improved Potency of Optimized LTBP-Complex-Specific Antibodies

LTBP complex-specific antibodies SR-AB10 and SR-AB13 were selected foran initial round of affinity maturation/optimization (i.e., H1/H2 CDRshuffling/diversification as described herein) and particular progenyantibodies (SR-AB14 and SR-AB15) were assessed for their ability toinhibit TGFβ activity using LN229 cells. For the assays depicted in FIG.12A and 12B, the following protocol was used, which is a modifiedversion of Assay II in Example 2. Materials:

MvLu1-CAGA12 cells (Clone 4A4)

LN229 cell line (high levels of endogenous αVβ8 integrin)

Costar white walled TC treated 96 well assay plate #3903

Greiner Bio-One High Binding white μclear 96 well assay plate #655094

Human Fibronectin (Corning #354008)

P200 multichannel pipet

P20, P200, and P1000 pipets with sterile filter tips for each

Sterile microfuge tubes and rack

Sterile reagent reservoirs

0.4% trypan blue

2 mL, 5 mL, 10 mL, and 25 mL sterile pipets

Tissue culture treated 100 mm or 150 mm plates

70%Ethanol

Opti-MEM reduced serum media (Life Tech #31985-070)

Lipofectamine 3000 (Life Tech #L3000015)

Bright-Glo luciferase assay reagent (Promega #E2620)

0.25% Trypsin +0.53mM EDTA

proTGFb1 expression plasmid, human

LTBP1S expression plasmid, human

Equipment:

PerkinElmer EnVision plate reader

TC hood

Bench top centrifuge

CO₂ incubator 37° C. 5% CO2

37° C. water/bead bath

Platform shaker

Microscope

Hemocytometer/countess

Definitions:

CAGA12 4A4 cells: Derivative of MvLul cells (Mink Lung EpithelialCells), stably transfected with CAGA12 synthetic promoter, drivingluciferase gene expression

DMEM-0.1%BSA: Assay media; base media is DMEM (Gibco Cat# 11995-065),media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin,and 4mM glutamine

D10: DMEM 10% FBS, P/S, 4mM glutamine, 1% NEAA, 1X GlutaMAX (Gibco Cat#35050061) CAGA12 (4A4) media: D10 +0.75ug/mL puromycin Procedure:

Always work in sterile biosafety cabinet, and sterile technique shouldbe used at all times.

Prepare all media, sterilize all materials, and move materials intobiosafety cabinet before starting.

Growth medium should be warmed to 37° C.

Use the reverse pipetting technique for almost all steps during thisassay.

Assays that rely on LTBP presentation of proTGFβ1 require pre-coating ofassay plates with fibronectin, for 4hrs—overnight prior to seeding cellsfor transfection.

On day—1, assay plates were coated with Fibronectin. A 1 mg/ml stocksolution of fibronectin was prepared in ultrapure water (sterile). Stocksolution was diluted in PBS to 19.2 μg/ml working solution and 50 μl wasadded to each well (3 μg/cm²). Plates were incubated at 37° C. and 5%CO₂ overnight.

On day 0, prior to cell seeding, assay plates were coated withFibronectin (LTBP over expression assay only). A lmg/ml stock solutionof fibronectin was prepared in ultrapure water (sterile). Stock solutionwas diluted in PBS to 19.2 μg/ml working solution and 50 μl was added toeach well (3 μg/cm²). Plates were incubated at 37° C. and 5% CO₂ for 4hours. Following coating, assay plates were washed manually withmultichannel pipette for 2 washes of 200 μl/well PBS. After final wash,plates were allowed to dry in the hood with lid off. LN229 cells werethen detached with trypsin and pelleted (spun for 5 min at 200×g). Thecell pellet was resuspended in D10 media and viable cells per mlcounted. Cells were diluted to 0.125e⁶ cells/ml and seeded 100 μl perwell (12,500 cells per well) in an assay plate. For CAGA12 cells to beused for assay on Day 2, cells passaged at a density of 1.5 million perT75 flask. Cultures were incubated at 37° C. and 5% CO₂.

On Day 1, LN229 cells were transfected. The manufacturer's protocol wasfollowed for transfection with Lipofectamine 3000 reagent. Briefly, thefollowing was diluted into OptiMEM I, for 5 μl per well: 0.1 μg DNA(proTGFβ31), optionally 0.1 μg LTBP1 DNA, 0.4 μl P3000, and up to 5 μlwith OptiMEM I. For FIG. 12A, 0.1 μg proTGFβ1 (human) DNA wastransfected alone, without LTBP DNA, to measure activation in thepresence of endogenous presenting molecules. For FIG. 12B, 0.1 μg LTBP1(human) DNA was co-transfected with the proTGFβ1 (human) DNA to measureactivation in the presence of overexpressed LTBP1.

A master mix of Lipofectamine3000 was made by diluting 0.41Lipofectamine3000 in OptiMEM I, up to 5 μl in OptiMEM I, per well.Diluted Lipofectamine3000 was added to the DNA mixture, mixed well bypipetting, and incubated at room temperature for 15 min. After theincubation, 10 μl of DNA:Lipofectamine3000 (2×5 μl) mixture was added toeach well. Plates were returned to the tissue culture incubator for −24hrs.

On day 2 the indicated antibodies and TGFβ reporter cells (CAGA12) wereadded to the wells. Antibodies were serially diluted into PBS, thenfurther diluted into assay media until 2× final concentration. Plateswere washed twice with assay media by aspirating (vacuum aspirator)followed by addition of 100 μl per well assay media. After second wash,assay media was replaced with 50 per well of 2X antibody. Cell plate wasreturned to the incubator for—15-20 min. CAGA12 (clone 4A4) cells weredetached with trypsin and pelleted (spun 5 min at 200×g.). Pellet wasresuspended in assay media and viable cells counted. Viable cells werediluted to 0.3e⁶cells/ml and seeded 50 μl per well (15,000 cells perwell). Cells were returned to the incubator.

On Day 3, the assay was read about 16-20 hours after the antibody and/orreporter cell addition. Bright-Glo reagent and test plate were allowedto come to room temperature before reading. The read settings on BioTekSynergy H1 were set to use TMLC_std protocol—this method has anauto-gain setting. Positive control wells were set for autoscale (high).100 μL of Bright-Glo reagent was added per well. Incubated for 2 minwith shaking, at room temperature, protected plate from light. Theluminescence was then detected on a plate reader. Results:

Data generated from this assay reflected TGFβ activity in cellsupernatants. FIG. 12A is a graph showing improved potency of SR-AB14(optimized SR-AB10) as measured by TGFβ activity. Notably, SR-AB14activity is similar to the context-independent antibody SR-AB 1. Thisassay was performed without overexpressing LTBP1 and thus measuresactivation of TGFβ in the presence of endogenous presenting molecules.FIG. 12B is a graph showing improved potency of SR-AB15 (optimizedSR-AB13) as measured by TGFβ activity. This assay was performed in thepresence of overexpressed human LTBP1-pro TGFβ1. Notably, SR-AB14activity is similar to the context-independent antibody SR-AB 1.

The assays depicted in FIGS. 12A and 12B were both performed in LN229cells, which express low LTBP1 mRNA, high LTBP3 mRNA, undetectable GARP,and undetectable LRRC33.

Example 8 Improved Affinity of Optimized LTBP-proTGF 1-SpecificAntibodies after CDR-H3 Mutagenesis

LTBP complex-specific antibodies from the first round of affinitymaturation/optimization (SR-AB14, SR-AB16, SR-AB17, SR-AB18, SR-AB19)were selected for a second round of affinity maturation/optimization(i.e., CDR-H3 mutagenesis as described herein) and particular progenyantibodies (SR-AB20, SR-AB21, SR-AB22, SR-AB23, SR-AB24, SR-AB25,SR-AB26, SR-AB27, SR-AB28, and SR-AB29) were assessed for their abilityto bind various proTGFβ1 constructs. Binding affinities of the optimizedantibodies were measured by Octet against human LTBP1-proTGFβ1, humanLTBP3-proTGFbl, mouse LTBP1-proTGFβ1, mouse LTBP3-proTGFbl, andGARP-proTGFβ1 complexes, essentially as described in Example 6. Inbrief, test antibodies were immobilized to the surface of anti-human Fccapture biosensors (AHC) (ForteBio®) and binding was then tested againstthe various TGFβ1 complexes at a single concentration of 100 nM toassess binding affinities. The antigens were allowed to associate for 3minutes followed by a 5-minute dissociation. Kinetics buffer (ForteBio®)was used throughout the experiment, and K_(D) was determined using a 1:1fitting model for each antibody antigen pair.

Table 8 presents the binding profile and affinity data for the indicatedLTBP complex-specific antibodies (IgGβ1-agly) determined by Octet.Notably, optimized antibodies SR-AB20, SR-AB21, SR-AB22, SR-AB23,SR-AB24, SR-AB25, SR-AB26, SR-AB27, SR-AB28, and SR-AB29 display singledigit nM affinity for both human LTBP1 and human LTBP3 complexed withproTGFβ1.

Additionally, none of the antibodies bound human GARP complexed withproTGFβ1 indicating that the antibodies are specific for LTBP complexes.

TABLE 8 ForteBio ForteBio ForteBio ForteBio ForteBio IgG K_(D) IgG K_(D)IgG K_(D) IgG K_(D) IgG K_(D) Hu LTBP1- Hu LTBP3- Mo LTBP1- Hu LTBP1- HuGARP- proTGFβ1 proTGFβ1 proTGFβ1 proTGFβ1 proTGFβ1 Ref Lineage (M) Avid(M) Avid (M) Avid (M) Avid (M) Avid SR-AB10 P.F. N.B. 1.42E−08 2.30E−08N.B. SR-AB16 SR-AB 10 3.44E−08 1.69E−08 3.29E−08 2.12E−08 N.B. SR-AB14SR-AB 10 2.44E−08 1.46E−08 2.04E−08 1.23E−08 N.B. SR-AB20 SR-AB163.93E−09 2.08E−09 6.46E−09 2.23E−09 N.B. SR-AB21 SR-AB16 6.78E−091.08E−09 1.06E−08 3.29E−08 N.B. SR-AB22 SR-AB14 1.88E−09 P.F. 2.43E−091.56E−09 P.F. SR-AB23 SR-AB14 5.98E−09 1.26E−09 8.38E−09 7.93E−09 N.B.SR-AB13 3.92E−08 3.59E−08 5.95E−08 N.B. N.B. SR-AB17 SR-AB13 6.16E−095.28E−09 8.83E−09 5.36E−08 N.B. SR-AB18 SR-AB13 9.27E−09 1.17E−081.45E−08 8.66E−08 N.B. SR-AB19 SR-AB13 1.10E−08 1.64E−08 1.59E−088.40E−08 N.B. SR-AB24 SR-AB17 1.74E−09 1.27E−09 2.07E−09 7.85E−08 N.B.SR-AB25 SR-AB17 2.37E−09 1.05E−09 2.92E−09 3.88E−08 N.B. SR-AB26 SR-AB172.73E−09 2.13E−09 3.73E−09 1.38E−08 N.B. SR-AB27 SR-AB18 2.70E−092.33E−09 3.89E−09 5.11E−07 N.B. SR-AB28 SR-AB18 2.84E−09 2.70E−094.52E−09 1.25E−08 N.B. SR-AB29 SR-AB19 4.23E−09 4.08E−09 7.22E−097.91E−08 N.B. P.F. = Poor Fit N.B. = Non-binder under conditions of thisassay

Example 9 Improved Affinity of Optimized LTBP-Complex-specificAntibodies after Light Chain Optimization Cycle 3

LTBP complex-specific antibodies from the second round of affinitymaturation/optimization were selected for a third round of affinitymaturation/optimization (i.e., light chain optimization as describedherein) and particular progeny antibodies, were assessed for theirability to bind various proTGFβ1 constructs.

Cycle 3 antibodies were generated by performing mutagenesis throughoutthe light chain CDR3 and performing a shuffle of premade sequences forthe light chain CDR1 and CDR2. Antibodies of interest were identifiedthrough yeast display utilizing both positive and negative selectionsfollowed by sequencing.

Binding affinities of the optimized antibodies were measured by Octetagainst human LTBP1-proTGFβ1, human LTBP3-proTGFβ1, mouseLTBP1-proTGFβ1, mouse LTBP3-proTGFβ1, and GARP-proTGFβ1 complexes,essentially as described in Example 6. In brief, test antibodies wereimmobilized to the surface of anti-human Fc capture biosensors (AHC)(ForteBio®) and binding was then tested against the various TGFβ1complexes at a single concentration of 100 nM to assess bindingaffinities. The antigens were allowed to associate for 3 minutesfollowed by a 5-minute dissociation. Kinetics buffer (ForteBio®) wasused throughout the experiment, and KD was determined using a 1:1fitting model for each antibody antigen pair.

Table 9 presents the binding profile and affinity data for the indicatedLTBP complex-specific antibodies (IgG1-agly) identified from the lightchain optimization cycle 3, determined by Octet. Notably, severaloptimized antibodies display single digit nM affinity for both humanLTBP1 and human LTBP3 complexed proTGFβ1. Additionally, as shown inTable 10, several antibodies did not bind human GARP complexed withproTGFβ1, under the same assay conditions, indicating that theantibodies are specific for LTBP complexes.

TABLE 9 Binding Profile and Affinity Data for Cycle 3 OptimizedAntibodies (N.B. = non-binder under conditions of this assay) ForteBioForteBio Fortebio Hu IgG K_(D) ForteBio ForteBio IgG K_(D) IgG K_(D)LTBP3- Hu Mo IgG K_(D) IgG K_(D) Hu LTBP1- Hu LTBP1- Hu LTBP1- Hu LTBP3-proTGFβ1 LTBP3- LTBP1- Mo LTBP3- Hu GARP- Optimization proTGFβ1 proTGFβ1proTGFβ1 proTGFβ1 Kon proTGFβ1 proTGFβ1 proTGFβ1 proTGFβ1 Ref Lineage(M) Avid Kon (1/Ms) Koff (1/s) (M) Avid (1/Ms) Koff (1/s) (M) Avid (M)Avid (M) Avid SR-AB30 SR-AB22 2.17E−10 2.77E+05 6.00E−05 1.56E−103.85E+05 6.00E−05 4.38E−10 4.21E−10 1.09E−08 SR-AB31 SR-AB22 4.12E−102.40E+05 9.89E−05 2.21E−10 3.08E+05 6.81E−05 6.82E−10 5.89E−10 1.12E−08SR-AB32 SR-AB22 3.22E−10 2.83E+05 9.14E−05 1.49E−10 4.02E+05 6.00E−054.85E−10 4.18E−10 1.09E−08 SR-AB33 SR-AB22 5.61E−10 3.02E+05 1.69E−042.72E−10 4.45E+05 1.21E−04 8.67E−10 4.78E−10 1.61E−08 SR-AB34 SR-AB222.31E−10 2.59E+05 6.00E−05 1.79E−10 3.72E+05 6.68E−05 4.17E−10 4.50E−107.35E−08 SR-AB35 SR-AB22 3.15E−10 3.42E+05 1.08E−04 1.41E−10 4.85E+056.85E−05 3.89E−10 3.98E−10 6.66E−09 SR-AB36 SR-AB22 2.54E−10 2.89E+057.34E−05 1.46E−10 4.10E+05 6.00E−05 3.35E−10 4.29E−10 9.55E−09 SR-AB37SR-AB22 3.28E−10 3.60E+05 1.18E−04 1.39E−10 5.45E+05 7.58E−05 3.84E−103.25E−10 3.61E−08 SR-AB38 SR-AB22 3.57E−10 3.27E+05 1.17E−04 1.45E−105.02E+05 7.26E−05 3.91E−10 3.31E−10 8.42E−09 SR-AB39 SR-AB22 2.05E−102.92E+05 6.00E−05 1.36E−10 4.41E+05 6.00E−05 2.32E−10 3.42E−10 3.89E−07SR-AB40 SR-AB23 4.01E−10 1.50E+05 6.00E−05 3.33E−10 1.82E+05 6.07E−051.08E−09 1.16E−09 N.B. SR-AB41 SR-AB23 2.96E−10 2.03E+05 6.00E−052.35E−10 2.55E+05 6.00E−05 4.04E−10 7.17E−10 7.64E−07 SR-AB42 SR-AB233.89E−10 1.54E+05 6.00E−05 2.59E−10 2.32E+05 6.00E−05 3.59E−10 1.05E−09N.B. SR-AB43 SR-AB23 6.49E−10 2.44E+05 1.59E−04 1.78E−10 3.37E+056.00E−05 7.31E−10 8.91E−10 N.B. SR-AB44 SR-AB23 2.14E−10 2.80E+056.00E−05 1.46E−10 4.10E+05 6.00E−05 2.10E−10 4.07E−10 2.12E−07 SR-AB45SR-AB23 1.93E−10 3.10E+05 6.00E−05 1.22E−10 4.91E+05 6.00E−05 1.93E−102.69E−10 1.95E−07 SR-AB62 SR-AB24 4.86E−10 1.40E+05 6.82E−05 3.68E−102.03E+05 7.47E−05 6.09E−10 3.37E−09 N.B. SR-AB63 SR-AB26 6.11E−101.18E+05 7.24E−05 4.94E−10 1.59E+05 7.84E−05 1.09E−09 2.00E−09 N.B.SR-AB64 SR-AB26 4.67E−10 4.71E−10 1.95E+05 9.18E−05 1.03E−09 2.85E−09N.B. Human IgG1 N.A. N.B. N.B. N.B. N.B. N.B. isotype control

TABLE 10 ForteBio VHCDR3 IgG K_(D) Hu GARP-proTGFβ1 Ref Lineage insolution (M) Avid SR-AB30 SR-AB10    1.09E−08 SR-AB31 SR-AB10   1.12E−08 SR-AB32 SR-AB10    1.09E−08 SR-AB33 SR-AB10    1.61E−08SR-AB34 SR-AB10    7.35E−08 SR-AB35 SR-AB10    6.66E−09 SR-AB36 SR-AB10   9.55E−09 SR-AB37 SR-AB10    3.61E−08 SR-AB38 SR-AB10    8.42E−09SR-AB39 SR-AB10    3.89E−07 SR-AB40 SR-AB10 N.B. SR-AB41 SR-AB107.63909E−07 SR-AB42 SR-AB10 N.B. SR-AB43 SR-AB10 N.B. SR-AB44 SR-AB10 2.1153E−07 SR-AB45 SR-AB10 1.95124E−07 SR-AB62 SR-AB13 N.B. SR-AB63SR-AB13 N.B. SR-AB64 SR-AB13 N.B. Human IgG1 isotype control N.A. N.B.N.B. = non-binder under conditions of this assay

FIG. 15 shows that affinity matured antibodies show specific binding tothe LTBP-proTGFβ1 complex. This experiment was performed at 200, 100,50, and 25 nM human GARP proTGFb1 and human LTBP1 proTGFbl. FIG. 15shows the 200 nM values, where the 0.lnm cutoff is used to determinewhat is and what is not meaningful binding. As shown in FIG. 15, forsome antibodies, binding to GARP-proTGFβ1 is meaningful only at veryhigh concentrations (200 nM), and some antibodies show no binding toGARP-proTGFβ1 even at that high concentration. Surface plasmon resonance(SPR)-based assays

A Biacore 8K system was employed to determine the monovalent bindingaffinity and the kinetic parameters for antigen binding of testantibodies. Association and dissociation kinetics of Fab fragmentsSR-AB42-HuFab, SR-AB63-HuFab and SR-AB43-HuFab to antigen complexes weremeasured, and resulting ka, kd and KD are provided below. Briefly, thebinding kinetics were evaluated by surface plasmon resonance usingBiacore 8K (GE Healthcare). Biotinylated capture antigens wereimmobilized to the chip (10 nM, —200 RU loading). A Biotin CAPturesensor chip was used to capture the biotinylated antigens. Fabs at 10,5, 2.5, 1.25, and 0.6 nM concentrations were injected over the capturedantigens. 0 nM was used as a reference. Affinities of LTBP antibodies toGARP and LRRC33 were confirmed using higher concentration of Fabs (100,50, 25, 12.5, 6.25 nM). 0 nM was used as a reference. Multi-cyclekinetics was employed where each analyte concentration was injected in aseparate cycle and the sensor chip surface was regenerated after eachcycle. All the assays were carried out in freshly prepared lxHBS-EP+buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween20, pH7.4). Data were fit globally to a 1:1 binding model to obtain thekinetic parameters. The sensorgram for 0 nM analyte concentration wasused as reference.

Results are shown in Tables 11-15 below. FIG. 23 and FIG. 24,respectively, show that SR-AB63 and SR-AB42 human Fabs showcontext-selective binding of novel antibodies for huLTBP1 proTGFβ1 andhuLTBP3 proTGFβ1.

TABLE 11 huLTBP1 proTGFβ1 ka (1/Ms) kd (1/s) KD (M) SR-AB42-HuFab*5.43E+05 1.05E−04 1.94E−10 SR-AB63-HuFab** 3.71E+05 3.82E−08 1.03E−13SR-AB43-HuFab 1.03E+06 2.84E−04 2.76E−10 SR-AB46-HuFab 2.47E+06 1.10E−044.44E−11 SR-AB47-HuFab 1.84E+06 1.36E−04 7.40E−11 SR-AB48-HuFab 1.76E+061.04E−04 5.93E−11 SR-AB49-HuFab 8.09E+05 1.38E−04 1.70E−10 SR-AB50-HuFab3.31E+06 1.82E−04 5.50E−11 SR-AB51-HuFab 2.55E+06 1.50E−04 5.89E−11SR-AB52-HuFab 2.50E+06 9.91E−05 3.96E−11 SR-AB53-HuFab 1.82E+06 1.28E−047.07E−11 SR-AB54-HuFab 1.80E+06 8.83E−05 4.91E−11 SR-AB55-HuFab 8.62E+051.13E−04 1.31E−10 SR-AB56-HuFab 2.58E+06 1.26E−04 4.89E−11 SR-AB57-HuFab1.35E+06 1.81E−04 1.33E−10 SR-AB58-HuFab 3.11E+06 1.62E−04 5.21E−11SR-AB59-HuFab 2.55E+06 1.30E−04 5.09E−11 SR-AB60-HuFab 2.35E+06 1.20E−045.10E−11 SR-AB61-HuFab 1.17E+06 1.55E−04 1.32E−10 *Average of fivereplicate experiments **Average of four replicate experiments

TABLE 12 huLTBP3 proTGFb1 ka (1/Ms) kd (1/s) KD (M) SR-AB42-HuFab4.05E+05 1.42E−04 3.50E−10 SR-AB63-HuFab 3.94E+05 4.99E−05 1.27E−10

TABLE 13 huGARP proTGFb1 ka (1/Ms) kd (1/s) KD (M) SR-AB42-HuFab1.55E+05 6.04E−03 3.89E−08 SR-AB63-HuFab 2.66E+03 9.30E−04 3.49E−07

TABLE 14 huLRRC33 proTGFb1 ka (1/Ms) kd (1/s) KD (M) SR-AB42-HuFab8.09E+04 3.53E−03 4.36E−08 SR-AB63-HuFab 5.00E+03 1.08E−03 2.16E−07

TABLE 15 Murine LTBP3 proTGFb1 ka (1/Ms) kd (1/s) KD (M) SR-AB42-HuFab*1.23E+08 3.97E−01 2.66E−09 SR-AB43-HuFab* 1.82E+09 6.37E+00 4.43E−09*Average of two replicate experiments

Example 10 Improved Potency of Optimized LTBP Complex-SpecificAntibodies after CDR-H3 Mutagenesis

LTBP complex-specific antibodies from the first round of affinitymaturation/optimization (i.e., SR-AB14, SR-AB16, SR-AB17, SR-AB18,SR-AB19) were selected for a second round of affinitymaturation/optimization (i.e., CDR-H3 mutagenesis as described herein)and particular progeny antibodies (SR-AB20, SR-AB21, SR-AB22, SR-AB23,SR-AB24, SR-AB25, SR-AB26, SR-AB27, SR-AB28, and SR-AB29) were assessedfor their ability to inhibit TGFβ activity using LN229 cells. For theassays depicted in FIG. 13A, 13B, 14A, and 14B the following protocolwas used, which is a modified version of Assay II in Example 2.

Materials:

MvLul-CAGA12 cells (Clone 4A4)

LN229 cell line (high levels of endogenous αVβ8 integrin)

Costar white walled TC treated 96 well assay plate #3903

Greiner Bio-One High Binding white uclear 96 well assay plate #655094

Human Fibronectin (Corning #354008)

P200 multichannel pipet

P20, P200, and P1000 pipets with sterile filter tips for each

Sterile microfuge tubes and rack

Sterile reagent reservoirs

0.4% trypan blue

2 mL, 5 mL, 10 mL, and 25 mL sterile pipets

Tissue culture treated 100 mm or 150 mm plates

70%Ethanol

Opti-MEM reduced serum media (Life Tech #31985-070)

Lipofectamine 3000 (Life Tech #L3000015)

Bright-Glo luciferase assay reagent (Promega #E2620)

0.25% Tryspin +0.53mM EDTA

proTGFb1 expression plasmid, human

proTGFb1 expression plasmid, mouse

LTBP1S expression plasmid, mouse

Equipment:

PerkinElmer EnVision plate reader

TC hood

Bench top centrifuge

CO₂ incubator 37C 5% CO2

37C water/bead bath

Platform shaker

Microscope

Hemocytometer/countess

Definitions:

CAGA12 4A4 cells: Derivative of MvLul cells (Mink Lung EpithelialCells), stably transfected with CAGA12 synthetic promoter, drivingluciferase gene expression

DMEM-0.1%BSA: Assay media; base media is DMEM (Gibco Cat# 11995-065),media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin,and 4mM glutamine

D10: DMEM 10% FBS, P/S, 4mM glutamine, 1% NEAA, 1X GlutaMAX (Gibco Cat#35050061)

CAGA12 (4A4) media: D10 +0.75ug/mL puromycin

Procedure:

Always work in sterile biosafety cabinet, and sterile technique shouldbe used at all times. Prepare all media, sterilize all materials, andmove materials into biosafety cabinet before starting. Growth mediumshould be warmed to 37° C.

Use the reverse pipetting technique for almost all steps during thisassay.

On day 0, prior to cell seeding, assay plates were coated withFibronectin (LTBP over expression assay only). A lmg/ml stock solutionof fibronectin was prepared in ultrapure water (sterile). Stock solutionwas diluted in PBS to 19.2ug/ml working solution and 50 μl was added toeach well (3 ug/cm²). Plates were incubated at 37° C. and 5% CO₂ for 4hours. Following coating, assay plates were washed manually withmultichannel pipette for 2 washes of 200 ul/well PBS. After final wash,plates were allowed to dry in the hood with lid off. LN229 cells werethen detached with trypsin and pelleted (spun for 5 min at 200×g). Thecell pellet was resuspended in D10 media and viable cells per mlcounted. Cells were diluted to 0.125e⁶ cells/ml and seeded 100 μl perwell (12,500 cells per well) in an assay plate. For CAGA12 cells to beused for assay on Day 2, cells passaged at a density of 1.5 million perT75 flask. Cultures were incubated at 37° C. and 5% CO₂.

On Day 1, LN229 cells were transfected. The manufacturer's protocol wasfollowed for transfection with Lipofectamine 3000 reagent. Briefly, thefollowing was diluted into OptiMEM I, for 5 μl per well: 0.1 μg proTGFβ1DNA combined with 0.1 μg presentation molecule DNA, and 0.4 μl P3000, upto 5 μl with OptiMEM I. For FIG. 13A and FIG. 14A, 0.1 μg human proTGFβ1DNA was transfected with 0.1ug empty vector, without LTBP DNA, tomeasure activation in the presence of endogenous presenting molecules.For FIG. 13B and FIG. 14B, 0.1 μg mouse LTBP1 DNA was co-transfectedwith the mouse proTGFβ1 DNA to measure activation in the presence ofoverexpressed LTBP1.

A master mix of Lipofectamine3000 was made by diluting 0.41Lipofectamine3000 in OptiMEM I, up to 5 μl in OptiMEM I, per well.Diluted Lipofectamine3000 was added to the DNA mixture, mixed well bypipetting, and incubated at room temperature for 15 min. After theincubation, 10 μul of DNA:Lipofectamine3000 (2×5 μl) mixture was addedto each well. Plates were returned to the tissue culture incubator for—24hrs.

On day 2 the indicated antibodies and TGFβ reporter cells (CAGA12) wereadded to the wells. Antibodies were serially diluted into PBS, thenfurther diluted into assay media until 2× final concentration. Plateswere washed twice with assay media by aspirating (vacuum aspirator)followed by addition of 100 μl per well assay media. After second wash,assay media was replaced with 50 μl per well of 2X antibody. Cell platewas returned to the incubator for—15-20 min. CAGA12 (clone 4A4) cellswere detached with trypsin and pelleted (spun 5 min at 200×g.). Pelletwas resuspended in assay media and viable cells counted. Viable cellswere diluted to 0.3e⁶cells/ml and seeded 50 μl per well (15,000 cellsper well). Cells were returned to the incubator.

On Day 3, the assay was read about 16-20 hours after the antibody and/orreporter cell addition. Bright-Glo reagent and test plate were allowedto come to room temperature before reading. 100 μL of Bright-Glo reagentwas added per well. Incubated for 2 min with shaking, at roomtemperature, protected plate from light. The luminescence was thendetected on a plate reader. Results:

Data generated from this assay reflected TGFβ activity in cellsupernatants. FIGS. 13A and 13B are graphs showing improved potency ofantibodies SR-AB20, SR-AB21, SR-AB22, and SR-AB23 (SR-AB10 familyantibodies) as measured by TGFβ activity. The FIG. 13A assay wasperformed without overexpressing LTBP1 and thus measures activation ofTGFβ in the presence of endogenous presenting molecules. The FIG. 13Bassay was performed with overexpressed murine LTBP1-proTGFβ1. Notably,antibodies SR-AB22 and SR-AB23 show activity that is similar to (orgreater than) the context-independent antibody SR-AB1 in both assays.

FIG. 14A and FIG. 14B are graphs showing improved potency of antibodiesSR-AB24, SR-AB25, SR-AB26, SR-AB27, SR-AB28, and SR-AB29 (SR-AB13 familyantibodies) as measured by TGFβ activity. The FIG. 14A assay wasperformed without overexpressing LTBP1 and thus measures activation ofTGFβ in the presence of endogenous presenting molecules. The FIG. 14Bassay was performed with overexpressed murine LTBP1-proTGFβ1. Notably,the optimized antibodies show activity that is greater than thecontext-independent antibody SR-AB1 in the LTBP1-proTGFβ1 overexpressioncell assay (FIG. 14B).

The assays depicted in FIG. 13A, FIG. 13B, FIG. 14A, and FIG. 14B wereall performed in LN229 cells, which express low LTBP1 mRNA, high LTBP3mRNA, undetectable GARP, and undetectable LRRC33.

Example 11 Improved Potency of Optimized LTBP Complex-SpecificAntibodies after Light Chain Mutagenesis

LTBP complex-specific antibodies from the third and fourth rounds ofaffinity maturation/optimization were assessed for their ability toinhibit TGFβ activity using LN229 cells. The assays were carried outusing the protocol described in Example 10.

To calculate inhibitory potency (IC50) values, raw luminescence valueswere normalized against wells treated with PBS only (vehicle).Normalized values were plotted using PRISM® software, and values werecalculated using 3-point non-linear regression. The Tables below showIC50 values (nM) for isoform-specific antibodies in two different assayformats: an endogenous human LTBP (hLTBP) assay and mouse LTBP assay(mLTBP). IC50 values were determined in the two assay formats for 4different antibody lineages: SR-AB22, SR-AB23, SR-AB24 and SR-AB26,shown in Tables 16-19.

TABLE 16 SR-AB22 lineage Endogenous mLTBP1 hLTBP assay assay Ref Ab 0.7682 SR-AB36 (cycle 3)  0.3 SR-AB34 (cycle 3)  0.9088 SR-AB30 (cycle3)  0.4038 SR-AB39 (cycle 3)  1.015 SR-AB34 (cycle 3)  0.4231 SR-AB36(cycle 3)  1.153 SR-AB35 (cycle 3)  0.4298 SR-AB31 (cycle 3)  1.261SR-AB31 (cycle 3)  0.4313 SR-AB35 (cycle 3)  1.343 SR-AB33 (cycle 3) 0.4491 SR-AB30 (cycle 3)  1.344 SR-AB32 (cycle 3)  0.4744 SR-AB31(cycle 3)  1.563 SR-AB39 (cycle 3)  0.4786 SR-AB37 (cycle 3)  1.93SR-AB37 (cycle 3)  0.5426 SR-AB32 (cycle 3)  2.223 SR-AB22 (cycle 2) 1.394 SR-AB10 (parent)  3.64 Ref Ab  1.571 SR-AB1  4.446 SR-AB1  6.381SR-AB22 (cycle 2)  4.618 SR-AB14 (cycle1) 12.59 SR-AB14 (cyclel) 51.65SR-AB10 (parent) 118.9

TABLE 17 SR-AB23 lineage Endogenous hLTBP mLTBP1 Ref Ab  0.7682 SR-AB45(cycle 3)  0.2638 SR-AB42 (cycle 3)  1.434 SR-AB42 (cycle 3)  0.2708SR-AB44 (cycle 3)  1.515 SR-AB44 (cycle 3)  0.3001 SR-AB41 (cycle 3) 1.542 SR-AB43 (cycle 3)  0.3189 SR-AB43 (cycle 3)  1.756 SR-AB40 (cycle3)  0.333 SR-AB40 (cycle 3)  2.105 SR-AB41 (cycle 3)  0.4086 SR-AB45(cycle 3)  2.247 SR-AB23 (cycle 2)  0.9458 SR-AB10 (parent)  3.64 Ref Ab 1.571 SR-AB1  4.446 SR-AB1  6.381 SR-AB23 (cycle 2)  6.043 SR-AB14(cycle1) 12.59 SR-AB14 (cyclel) 51.65 SR-AB10 (parent) 118.9

TABLE 18 SR-AB24 lineage Endogenous hLTBP mLTBPI Ref Ab  0.7682 SR-AB62(cycle 3)  0.3861 SR-AB1  4.446 SR-AB24 (cycle 2)  1.313 SR-AB13(parent)  4.825 Ref Ab  1.571 SR-AB62 (cycle 3)  8.683 SR-AB17 (cycle 1) 4.038 SR-AB24 (cycle 2)  9.599 SR-AB1  6.381 SR-AB17 (cycle 1) 24.14SR-AB13 (parent) 25.44

TABLE 19 SR-AB26 lineage Endogenous hLTBP mLTBP1 SR-AB1  0.7682 SR-AB63(cycle 3)  0.2075 SR-AB63 (cycle 3)  1.777 SR-AB64 (cycle 3)  0.2683SR-AB64 (cycle 3)  3.12 SR-AB26 (cycle 2)  1.172 SR-AB1  4.446 Ref Ab 1.571 SR-AB13 (parent)  4.825 SR-AB17 (cycle 1)  4.038 SR-AB26 (cycle2)  8.742 SR-AB1  6.381 SR-AB17 (cycle 1) 24.14 SR-AB13 (parent) 25.44

Data generated from this assay reflected TGFβ activity in cellsupernatants. As shown in FIG. 16, the functional activity (potency) ofLTBP complex specific antibodies improved in each cycle of affinitymaturation. As shown in FIG. 16, improvement from each cycle of affinitymaturation resulted in activity exceeding the context-independentreference antibody (Ref Ab), a potent context-independent inhibitor oflatent TGFβ1. The assays described above were all performed in LN229cells transiently transfected with murine LTBP1 and murine proTGFβ1.

For the results shown in Table 20, the assay was carried out using theprotocol described in Example 10, but in this assay Fabs were utilized.

TABLE 20 Antibody IC50, nM RefAb (hlgG4)  0.7676 RefAb (Fab)  3.607SR-AB47 Fab  0.1861 SR-AB59 Fab  0.1947 SR-Ab56 Fab  0.4393 SR-AB52 Fab 0.4589 SR-AB61 Fab  0.4699 SR-AB41 Fab  0.4861 SR-AB46 Fab  0.4946SR-AB48 Fab  0.5233 SR-AB51 Fab  0.5354 SR-AB55 Fab  0.5363 SR-AB42 Fab 0.6675 SR-AB60 Fab  0.742 SR-AB57 Fab  0.7754 SR-AB49 Fab  1.024SR-AB43 Fab  1.077 SR-AB53 Fab  1.181 SR-AB58 Fab  4.856 SR-AB54 Fab 5.87 SR-AB23 Fab  8.533 SR-AB50 Fab 10.97

Example 12 Developability

Antibodies that exhibit favorable biophysical properties have anincreased success rate in development. LTBP complex-specific antibodiesfrom the third and fourth rounds of affinity maturation/optimizationwere assessed for aggregation propensity and polyspecificity . FIG. 17shows the results of an enzyme-linked immunosorbent assay (ELISA)showing SR-AB binding to baculovirus (BV) particles. FIG. 18 shows theresults of affinity-capture Self-interaction Nanoparticle Spectroscopy(AC-SINS) Assay. This assay tests how likely an antibody is to interactwith itself. It uses gold nanoparticles that are coated with anti-Fcantibodies. When a dilute solution of antibodies is added, they rapidlybecome immobilized on the Fc-coated gold beads. If these antibodiessubsequently interact with one another, it leads to shorter interatomicdistances and an increase in the plasmon wavelength that can be detectedby spectroscopy.

These results show that the candidate antibodies demonstrate favorabledevelopability properties, as evidenced by a plasmon shift less than 5nM in the AC-SINS assay and a BV ELISA signal less than 5 fold of thenegative control.

Example 13 Inhibition of TGFβ Signaling by LTBP Complex-SpecificAntibodies in Choline-Deficient High Fat Diet (CDHFD) Model of MouseNASH

The Choline deficient high fat diet (CDHFD) is an establisheddiet-induced model of Non-Alcoholic steatohepatitis (NASH). In thismodel, male C57BL/6J mice are fed a choline deficient, 0.1% Methionine,high fat diet for 12 weeks. Three to six weeks after the start of theCDHFD, activation of pro-fibrotic processes can be detected throughincreased expression of a-smooth muscle actin (a-SMA) protein, a markerfor activation of hepatic stellate cells, and elevated tissuehydroxyproline content reflecting increased collagen synthesis anddeposition (Matsumoto et. al, Int J Exp Pathol. 2013 Apr; 94(2):93-103).

LTBP complex-specific antibodies were tested for their ability toinhibit and/or reduce the extent of liver fibrosis in mice in the CDHFDmodel as follows. An outline of the study is shown below in Table 21.

TABLE 21 Dose Group Diet Lineage Ab (mlgG1) (mg/kg) Frequency Takedown N1 Regular Chow NA NA NA  4 weeks 5 2 CDAA/HFD NA NA NA  4 weeks 8 3Regular Chow HuNeg 15 2× weekly 12 weeks 5 4 CDAA/HFD HuNeg 15 2× weekly12 weeks 10 3 CDAA/HFD Ref Ab 15 2× weekly 12 weeks 10 4 CDAA/HFDSR-AB23 SR-AB42 15 2× weekly 12 weeks 10 5 CDAA/HFD SR-AB22 SR-AB31 152× weekly 12 weeks 10

Animals in the test cohorts were on the CDHFD for the duration of thestudy. A separate cohort of mice (Group 1) were administered a regularchow diet as a study control. Antibodies SR-AB42 and SR-AB31 wereadministered to mice by intraperitoneal (i.p.) injection beginning at 4weeks post start of the CDHFD Animals were dosed at antibody testconcentrations 15mg/kg twice a week (i.e., 30 mg/kg/week) for a testduration of 8 weeks (i.e., weeks 4 through 12). A mouse IgG1 isotypeantibody was used as a negative control at 15mg/kg twice weekly (i.e.,30 mg/kg/week). Ref Ab was used as a reference antibody due to it beinga potent context-independent inhibitor of TGFβ1. Following 8 weeks ofdosing, animals were sacrificed and livers were collected for analysis.Endpoint readout was hydroxyproline content.

Hydroxyproline Content in CDHFD Liver Fibrosis Model

Hydroxyproline, produced by hydroxylation of the amino acid proline, isa signature amino acid for major component of fibrillar collagens, andcomprises approximately 13.5% of the protein. Hydroxyproline acts as animportant diagnostic indicator of the severity of fibrosis. Animals feda CDHFD show an increase in hydroxyproline (HYP) in liver tissue. Asshown in FIG. 19, treatment with SR-AB42 and SR-AB31 inhibited theincrease in HYP (μg/mg tissue) in liver tissue in animals on CDHFD.

Example 14 Inhibition of TGFβ Signaling by LTBP Complex-SpecificAntibodies in a Genetic Model of Alport Syndrome

The murine Col4a3 −/− model is an established genetic model of autosomalrecessive Alport5 syndrome. Alport mice lack a functional collagen 4A3gene (Col4A3-/-) and therefore cannot form normal type IV collagentrimers, which require a3, a4, and a5 chains. Col4a3−/− mice developfibrosis in the kidney consistent with renal fibrosis in human patients,including interstitial fibrosis and tubular atrophy, and Col4a3−/− micedevelop end-stage renal disease (ESRD) between 8 and 30 weeks of age,depending on the genetic background of the mouse. The structural andfunctional manifestation of renal pathology in Col4a3−/− mice, combinedwith the progression to ESRD make Col4a3−/− mice an ideal model tounderstand kidney fibrosis. Previous reports point to the importance ofthe TGFβ signaling pathway in this process, and treatment with either aninhibitor of αVβ6 integrin, a known activator of TGFβ1 and TGFβ3, orwith a TGFβ ligand trap has been reported to prevent renal fibrosis andinflammation in Alport mice (Hahm et al. (2007) The American Journal ofPathology, 170(1): 110-125).

LTBP complex-specific antibodies were tested for their ability toinhibit and/or reduce the renal fibroses in Alport mice as follows. Anoutline of the study is shown below in Table 22.

TABLE 22 Group Genotype Ab (hIgG4) Dose (mg/kg) Frequency N 1 Het HuNeg30 1  6 2 KO HuNeg 30 1 10 3 KO Ref Ab 30 1 10 4 KO SR-AB42 30 1 10 5 KOSR-AB63 30 1 10

F1 offspring from Col4a3 +/− males on a 129/Sv genetic backgroundcrossed to Col4a3 +/− females on a C57BL/6 genetic background wereemployed for the study. These mice typically exhibit proteinuria by 4-5weeks old and typically progress to ESRD by 13-15 weeks old, providing agood therapeutic window for testing efficacy of treatment. 11 week oldCol4a3−/− mice were dosed with 30 mg/kg SR-AB42 and SR-AB63intraperitoneally (i.p.) 48 hours prior to animal sacrifice and kidneycollection. Ref Ab was used as a reference antibody due to it being apotent context-independent inhibitor of TGFβ1 activation. pSMAD Analysisin Alport Kidney Model

It is well documented that TGFβ receptor activation leads to adownstream signaling cascade of intracellular events, includingphosphorylation of Smad2/3. Therefore, the ability of SR-AB42 andSR-AB63 treatment to inhibit TGFβ signaling was assessed in kidneylysate samples by measuring relative phosphorylation levels of Smad2/3as assayed by ELISA (Cell Signaling Technologies) according to themanufacturer's instructions. FIG. 20A is a graph showing relative ratiosof phosphorylated versus total (phosphorylated and unphospohrylated)Smad2/3 (pSMAD2/3:tSMAD2/3) in an Alport mouse model. FIG. 20B is agraph showing the amount of phosphorylated SMAD2/3 (pSMAD2/3) asdetermined by ELISA and FIG. 20C is a graph showing the amount of totalSMAD2/2 (tSMAD2/3) protein as determined by ELISA. As shown by FIG. 20Band FIG. 20C, reduction of pSMAD is contributing to the change in ratioshown in FIG. 20A. A single dose of SR-AB42 or SR-AB63 was sufficient tosignificantly inhibit pSmad2/3 signaling in whole kidney lysates,demonstrating efficient target engagement by SR-AB42 and SR-AB63 andthat LTBP complex-specific inhibition in the Alport model reduces pSmadlevels.

Example 15 Determination of proTGβ1 C4S binding

A modified proTGFβ1 complex (proTGFβ1 C4S) in which the cysteine residueat position 4 of the pro-domain has been substituted with a serineresidue (described in WO 2014/182676) was used to assess antibodybinding to C4S antigen by Octet. Cycle 2 antibodies were immobilized andtested against 3 antigens. As shown in the Tables below, robust antibodybinding to LTBP-1 proTGFβ1 antigen (Table 23) and proTGFβ1C4S antigenwas observed (Table 24), while binding to LRRC33 proTGFβ1 antigen wasnot observed (Table 25).

TABLE 23 Binding to human LTBP-1 proTGFb1 Loading Response Sample IDSample ID (nm) human LTBP-1 proTGFb1 SR-AB14 0.3197 human LTBP-1proTGFb1 SR-AB20 0.4132 human LTBP-1 proTGFb1 SR-AB21 0.3196 humanLTBP-1 proTGFb1 SR-AB22 0.4317 human LTBP-1 proTGFb1 SR-AB23 0.3449human LTBP-1 proTGFb1 SR-AB17 0.3036 human LTBP-1 proTGFb1 SR-AB240.3796 human LTBP-1 proTGFb1 SR-AB25 0.3085 human LTBP-1 proTGFb1SR-AB26 0.2953 human LTBP-1 proTGFb1 SR-AB27 0.3717 human LTBP-1proTGFb1 SR-AB28 0.4046 human LTBP-1 proTGFb1 SR-AB29 0.3242 humanLTBP-1 proTGFb1 HuNeg −0.0007 human LTBP-1 proTGFb1 SR-AB16 0.2594 humanLTBP-1 proTGFb1 SR-AB1 0.4311

TABLE 24 Binding to human TGFb1 C4S Loading Sample Response Sample ID ID(nm) human TGFb1 C4S SR-AB14 0.2002 human TGFb1 C4S SR-AB20 0.2891 humanTGFb1 C4S SR-AB21 0.1812 human TGFb1 C4S SR-AB22 0.2937 human TGFb1 C4SSR-AB23 0.2263 human TGFb1 C4S SR-AB17 0.2835 human TGFb1 C4S SR-AB240.3846 human TGFb1 C4S SR-AB25 0.329 human TGFb1 C4S SR-AB26 0.3764human TGFb1 C4S SR-AB27 0.3814 human TGFb1 C4S SR-AB28 0.3605 humanTGFb1 C4S SR-AB29 0.3334 human TGFb1 C4S HuNeg 0.001 human TGFb1 C4SSR-AB16 0.1454 human TGFb1 C4S SR-AB1 0.3604

TABLE 25 Binding to human LRRC33 proTGFb1 Loading Response Sample IDSample ID (nm) human LRRC33 proTGFb1 SR-AB14 0.03 human LRRC33 proTGFb1SR-AB20 0.0894 human LRRC33 proTGFb1 SR-AB21 0.0141 human LRRC33proTGFb1 SR-AB22 0.1329 human LRRC33 proTGFb1 SR-AB23 0.0233 humanLRRC33 proTGFb1 SR-AB17 0.0043 human LRRC33 proTGFb1 SR-AB24 0.0394human LRRC33 proTGFb1 SR-AB25 0.024 human LRRC33 proTGFb1 SR-AB26 0.0208human LRRC33 proTGFb1 SR-AB27 0.0317 human LRRC33 proTGFb1 SR-AB28 0.037human LRRC33 proTGFb1 SR-AB29 0.0288 human LRRC33 proTGFb1 HuNeg 0.0023human LRRC33 proTGFb1 SR-AB16 0.0161 human LRRC33 proTGFb1 SR-AB1 0.4006

Example 16 Cycle 3 Lead Antibodies Show No Inhibition in TGF/β3 Assay

FIG. 21 is a graph showing that the lead cycle 3 antibodies show noinhibition in the LTBP-TGFβ33 assay. The TGFβ3 assay is performedsimilarly to the assay in Example 10, but proTGFβ3 is transfectedinstead of proTGFβ1. No LTBP construct is transfected as this assayrelies on the endogenous presentation molecules in the LN229 cell line

Example 17 Pro Fibrotic Effects of TGF/13-Selective Activation Inhibitorin Liver Fibrosis Model

Liver expresses both TGFβ1 and TGFβ3. To investigate whether inhibitionof both isoforms in the CDHFD model would further mitigate fibrosis ofthe liver, CDHFD mice were treated with a potent TGFβ1-selectiveactivation inhibitor (which inhibits activation of TGFβ1 in the contextof human LTBP1 and LTBP3 complexes) and a TGFβ3-selective activationinhibitor, either alone or in combination. In mice treated with TGFβ3inhibitor, exacerbation of the disease was observed, as evidenced by PSRanalysis and additional histopathology analyses (FIG. 22). At 12 weeks,e.g., end of the study, animals that received the TGFβ1-selectiveactivation inhibitor showed significantly less fibrosis as compared toIgG-treated animals (negative control). By contrast, animals treatedwith TGFβ3-selective inhibitor showed significantly more fibrosis withapproximately 12% PSR positive area Animals that received a combinationof both TGFβ3-selective inhibitor and TGFβ1-selective inhibitor showedan intermediate level of fibrosis, indicating that antifibrotic effectsof the TGFβ1-selective activation inhibitor is being mitigated by TGFβ3inhibition.

Example 18 Improved Affinity of Optimized LTBP-Complex-specificAntibodies after Light Chain Optimization Cycle 4

LTBP complex-specific antibodies from the third round of affinitymaturation/optimization were selected for a fourth round of affinitymaturation/optimization (i.e., light chain optimization as describedherein) and particular progeny antibodies, were assessed for theirability to bind various proTGFβ1 constructs.

Materials:

96 well plates—Greinerbio-one REF 650101

Microplate Foils-GE Healthcare—CAT# 28-9758-16

Biotin CAPture Kit GE Healthcare product number 28920233

Biacore running buffer—supplied as a 20x solution, diluted in Milli-Qwater, TEKnova

CAT# H8022

Method:

These experiments were performed using the Biacore 8K using the method“multi-cycle kinetics/affinity using Biotin CAPture kit.”

Data collection rate was set to 10 Hz and the Biotin CAPture step wasperformed for 300 seconds with a flow rate of 2 μL/minute. The BiotinCAPture reagent was used after a 1:5 dilution in 1×Biacore runningbuffer. Biotinylated ligands (the various complexes of TGFβ31) wereutilized at a concentration of 10 nM and were immobilized to the sensorchip for 180 seconds with a flow rate of 10 μL/minute. The analytes,which are each of the Fabs, were tested at 10, 5, 2.5, 1.25, and 0.625nM with a 0 nM control included. Analyte contact time was 120 secondsand the dissociation time was 900 seconds. The regeneration cycleutilized was 120 seconds with a flow rate of 10 μL/minute.

The evaluation method used was “multi-cycle affinity using capture” anda 1:1 binding model was used. For every Fab tested against a givenbiotinylated antigen, the Rmax was set to equal the Rmax of anaffinity-matured version of Reference Ab with significantly slowerbinding off-rate and therefore higher affinity binding to that sameantigen in the same experiment. Global fits were utilized for every KDdetermination. The KD values are shown in Table 26 below.

TABLE 26 HuLTBP1- HuLTBP3- HuGARP- HuLRRC33- ProTGFb1 ProTGFb1 ProTGFb1ProTGFb1 SR-AB42-HuFab 0.381 0.57 144.614 134.249 SR-AB47-HuFab 0.2170.33  6.496  6.663 SR-AB49-HuFab 0.319 0.58  6.551  7.704 Ref Ab-HuFab0.077 0.11  0.202  0.154 MuLTBP1- MuLTBP3- MuGARP- MuLRRC33- ProTGFb1ProTGFb1 ProTGFb1 ProTGFb1 SR-AB42-HuFab 0.611 0.68  29.102 109.989SR-AB47-HuFab 0.198 0.40  1.950  9.164 SR-AB49HuFab 0.379 0.65  2.820 6.945 Ref Ab-HuFab 0.078 0.09  0.264  0.107

Table 27 shows the monovalent half-binding-times (T½). The monovalenthalf-binding-time (T½) value was at least 45 minutes for each ofhLTBP1-proTGFβ1 and hLTBP3-proTGFβ1 complexes. The monovalent T½ valuewas less than 5 minutes for each of hGARP-proTGFβ1 and hLRRC33-proTGFβ1complexes, as measured by SPR.

TABLE 27 Monovalent Half-Binding Times (T 1/2) LTBP-SR- KD T_(1/2) AB42HuFab (nM) Binding Hu LTBP1-ProTGFβ1  0.38 1.7 hours Hu LTBP3-ProTGFβ1 0.57 1.7 hours Hu GARP-ProTGFβ1 144 1.9 min Hu LRRC33-ProTGFβ1 134 4.3min Mu LTBP1-ProTGFβ1  0.6 1.1 hours Mu LTBP3-ProTGFβ1  0.68 1.2 hoursMu GARP-ProTGFβ1  29 5.1 min Mu LRRC33-ProTGFβ1 110 7.4 min

Embodiments

-   The present invention encompasses various embodiments. Non-limiting    examples are listed below:-   1. An antibody, or antigen-binding fragment thereof, comprising at    least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:94 (SEQ ID NO: 94 comprising these substitutions isdisclosed as SEQ ID NO: 399), with the proviso that, optionally:

-   -   i. the threonine residue at position 2 of SEQ ID NO:94 may be        substituted with an alanine;    -   ii. the asparagine residue at position 4 of SEQ ID NO:94 may be        substituted with an alanine, tyrosine, aspartate, serine,        arginine, or histidine;    -   iii. the asparagine residue at position 5 of SEQ ID NO:94 may be        substituted with a glutamine, serine, glycine, lysine,        glutamate, arginine, or histidine;    -   iv. the tyrosine residue at position 6 of SEQ ID NO:94 may be        substituted with a arginine;    -   v. the proline residue at position 7 of SEQ ID NO:94 may be        substituted with a glycine, alanine, leucine, serine,        asparagine, valine, aspartate, or glutamine;    -   vi. the isoleucine residue at position 8 of SEQ ID NO:94 may be        substituted with a methionine or leucine; and/or,    -   vii. the histidine residue at position 9 of SEQ ID NO:94 may be        substituted with a phenylalanine, tyrosine, asparagine, or        serine;

b) CDR-H2: SEQ ID NO:95, optionally comprising one or more amino acidchanges;

c) CDR-H3: SEQ ID NO:96, optionally comprising one or more amino acidchanges;

d) CDR-L1: SEQ ID NO:97, optionally comprising one or more amino acidchanges;

e) CDR-L2: SEQ ID NO:98, optionally comprising one or more amino acidchanges; and,

f) CDR-L3: SEQ ID NO:99, optionally comprising one or more amino acidchanges.

-   2. The antibody, or antigen-binding fragment thereof, according to    embodiment 1, comprising at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTF(X₁)(X₂)YVMH, wherein,optionally: X₁ is S or R; and X₂ is G or S (SEQ ID NO: 392);

b) CDR-H2 comprising the amino acid sequence (X₁)ISHEG(X₂)(X₃)KYYADSVKG,wherein, optionally: X₁ is V or S; X₂ is S or G; and X₃ is F or L (SEQID NO: 393); and

c) CDR-H3 comprising the amino acid sequence(X₁)(X₂)P(X₃)(X₄)(X₅)(X₆)RRGG(X₇) (X₈)(X₉), wherein, optionally: X₁ is Aor V; X₂ is R, V, G or K; X₃ is R, H or L; X₄ is I, V or G; X₅ is A, S,or L; X₆ is A or V; X₇ is F or Y; X₈ is D, G, R, or S; and, X₉ is Y, G,R, L, V, A or K (SEQ ID NO: 394).

d) CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one ormore amino acid changes;

e) CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one ormore amino acid changes; and

f) CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one ormore amino acid changes.

-   3. The antibody, or antigen-binding fragment thereof, according to    embodiment 2, wherein:

a) within CDR-H2; X₂ is S; and

b) within CDR-H3; X₁ is A; X₂ is R or V; X₃ is R; X₄ is I; X₅ is A or L;X₆ is A; X₇ is F; X₈ is G; and X₉ and Y.

-   3.1 The antibody, or antigen-binding fragment thereof, according to    embodiment 3, wherein one or more of the CDR-L1, CDR-L2, and CDR-L3    is/are affinity matured and/or optimized by CDR diversification    and/or mutagenesis.-   4. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, comprises a heavy chain variable    region having an amino acid sequence that is at least 90% identical    to SEQ ID NO: SEQ ID NO: 88; and/or,    -   wherein the antibody comprises a variable light chain variable        region having an amino acid sequence that is at least 90%        identical to SEQ ID NO: 89.-   5. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, comprising:    -   a heavy chain variable region having an amino acid sequence that        is at least 90% identical to SEQ ID NO: 88; and    -   a light chain variable region having an amino acid sequence that        is at least 90% identical to SEQ ID NO: 89.-   6. An antibody, or antigen-binding fragment thereof, comprising at    least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:100 (SEQ ID NO: 100 comprising these substitutionsis disclosed as SEQ ID NO: 400), with the proviso that, optionally:

-   -   i. the serine residue at position 4 of SEQ ID NO:100 may be        substituted with a histidine;    -   ii. the serine residue at position 7 of SEQ ID NO:100 may be        substituted with an alanine or glycine; and/or,    -   iii. the glycine residue at position 11 of SEQ ID NO:100 may be        substituted with a threonine, serine, histidine, leucine,        isoleucine, asparagine, valine, or alanine;

b) CDR-H2: SEQ ID NO:101 (SEQ ID NO: 101 comprising these substitutionsis disclosed as SEQ ID NO: 401), with the proviso that, optionally:

-   -   i. the serine residue at position 3 of SEQ ID NO:101 may be        substituted with an alanine;    -   ii. the glycine residue at position 6 of SEQ ID NO:101 may be        substituted with an alanine or serine; and/or,    -   iii. the serine residue at position 7 of SEQ ID NO:101 may be        substituted with a threonine;

c) CDR-H3: SEQ ID NO:102, optionally comprising one or more amino acidchanges;

d) CDR-L1: SEQ ID NO:103, optionally comprising one or more amino acidchanges;

e) CDR-L2: SEQ ID NO:104, optionally comprising one or more amino acidchanges; and,

f) CDR-L3: SEQ ID NO:105, optionally comprising one or more amino acidchanges.

-   7. The antibody, or antigen-binding fragment thereof, according to    embodiment 6, comprising at least three of the following six CDRs:

g) CDR-H1 comprising the amino acid sequence G(X₁)I(X₂)S(X₃)SYYW(X₄),wherein, optionally: X₁ is S or P; X₂ is S, H or R; X₃ is S or G; and,X₄ is G, I, N or V (SEQ ID NO: 395);

h) CDR-H2 comprising the amino acid sequence SISYSA(X₁)TYYNPSLKS,wherein, optionally: X₁ is S or T (SEQ ID NO: 396);

i) CDR-H3 comprising the amino acid sequence(X₁)(X₂)D(X₃)(X₄)Y(X₅)(X₆)(X₇)(X₈)G(X₉) (X₁₀)(X₁₁), wherein, optionally:X₁ is A or V; X₂ is R, S or G, X₃ is P, Y, R, V, I, H, T or E; X₄ is S,D, E or N; X₅ is D, A or T; X₆ is S, G, T or A; X₇ is I, A, R, Q, or V;X₈ is A, E, K, G or T; X₉ is M or I; X₁₀ is D, L, Q, V, N or G; and, X₁i is V, R, N, E or K (SEQ ID NO: 397);

-   -   j) CDR-Ll: SEQ ID NO:103, optionally comprising one or more        amino acid changes;    -   k) CDR-L2: SEQ ID NO: 104, optionally comprising one or more        amino acid changes; and    -   l) CDR-L3: SEQ ID NO: 105, optionally comprising one or more        amino acid changes.

8. The antibody, or antigen-binding fragment thereof, according toembodiment 7, wherein:

-   -   g) within CDR-H1: X₂ is H or R; X₃ is S; and, X₄ is G, I or N;        and    -   h) within CDR-H3: X₁ is A; X₃ is P or V; X₄ is S; X₅ is D; X₆ is        S or A; X₇ is A, R, I or V; X₈ is A; X₉ is M; X₁ o is D, Q, or        G; and, X₁ i is V or R.

8.1 The antibody, or antigen-binding fragment thereof, according toembodiment 8, wherein one or more of the CDR-L1, CDR-L2, and CDR-L3is/are affinity matured and/or optimized by CDR diversification and/ormutagenesis.

9. The antibody, or antigen-binding fragment thereof, according to anyone of embodiments 6-8.1, comprising at least three CDRs selected fromthe following, optionally comprising one or more amino acid changes foreach of the CDRs:

CDR-H1: SEQ ID NO:100;

CDR-H2: SEQ ID NO:101;

CDR-H3: SEQ ID NO:102;

CDR-Ll: SEQ ID NO:103;

CDR-L2: SEQ ID NO:104; and,

CDR-L3: SEQ ID NO:105.

-   10. The antibody, or antigen-binding fragment thereof, according to    one of embodiments 6-9, comprising a variable heavy chain region and    a variable light chain region, wherein:

i) the variable heavy chain region comprises an amino acid sequence thatis at least 90% identical to SEQ ID NO: SEQ ID NO: 106; and/or,

ii) the variable light chain variable region comprises an amino acidsequence that is at least 90% identical to SEQ ID NO: 107.

-   11. The antibody, or antigen-binding fragment thereof, according to    one of embodiments 6-9, wherein the antibody or antigen-binding    portion thereof, comprises:

i) a variable heavy chain region having an amino acid sequence that isat least 90% identical to SEQ ID NO: 106; and,

ii) a light chain variable region having an amino acid sequence that isat least 90% identical to SEQ ID NO: 107.

-   12. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, comprising all six CDRs.-   12.1 The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the one or more amino    acid changes comprises one amino acid change.-   12.2 The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the one or more amino    acid changes comprises up to two amino acid changes.-   12.3 The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the one or more amino    acid changes comprises up to three amino acid changes.-   12.4 The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the one or more amino    acid changes comprises up to four amino acid changes.-   12.5 The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the one or more amino    acid changes comprises up to five amino acid changes.-   12.6 The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the one or more amino    acid changes comprises up to six amino acid changes.-   12.7 The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the one or more amino    acid changes comprises up to seven amino acid changes.-   12.8 The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein any one of the CDRs    comprise no amino acid changes.-   13. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, specifically binds a human    LTBP1-proTGFβ complex.-   14. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, specifically binds a human    LTBP3-proTGFβ complex.-   15. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, does not bind a human GARP-proTGFβ    complex.-   16. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, specifically binds a human    LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex, and does    not bind a human GARP-proTGFβ complex.-   17. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof does not bind mature TGFβ1, mature    TGFβ2 or mature TGFβ3.-   18. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, is a fully human or humanized    antibody.-   19. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, is an isolated antibody, or    antigen-binding fragment thereof.-   20. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, does not compete with any one of    the antibodies SR-Ab1, SR-Ab2 or SR-Ab13 for binding to a human    LTBP1-proTGFβ1 complex.-   21. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, does not compete with any one of    the antibodies SR-Ab1, SR-Ab2 or SR-Ab10 for binding to a human    LTBP1-proTGFβ1 complex.

22. The antibody, or antigen-binding fragment thereof, according to anyone of the preceding embodiments, wherein the antibody is an IgG4 orIgG1 subtype.

23. The antibody, or antigen-binding fragment thereof, according to anyone of the preceding embodiments, wherein the antibody is specific forhuman LTBP1-TGFβ1 complex.

24. The antibody, or antigen-binding fragment thereof, according to anyone of the preceding embodiments, wherein the antibody is specific forhuman LTBP3-TGFβ1 complex.

-   25. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody is    specific for human LTBP1-TGFβ1 complex and human LTBP3-TGFβ1    complex.-   26. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody does not    bind a human GARP-TGFβ1 complex or GARP-TGFβ3 complex.-   27. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, binds a human LTBP1-proTGFβ1    complex with a K_(D) of <50 nM as measured by Bio-Layer    Interferometry (BLI).-   28. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, binds a human LTBP3-proTGFβ1    complex with a K_(D) of <50 nM as measured by Bio-Layer    Interferometry (BLI).-   29. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, binds a human LTBP1-proTGFβ1    complex with a K_(D) of <10 nM as measured by Bio-Layer    Interferometry (BLI).-   30. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, binds a human LTBP3-proTGFβ1    complex with a K_(D) of <10 nM as measured by Bio-Layer    Interferometry (BLI).-   31. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 27-30, wherein the antibody, or    antigen-binding fragment thereof, is cross-reactive with mouse    LTBP1-proTGFβ1.-   32. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 27-30, wherein the antibody, or    antigen-binding fragment thereof, is cross-reactive with mouse    LTBP3-proTGFβ1.-   33. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 27-30, wherein the antibody, or    antigen-binding fragment thereof, is cross-reactive with both mouse    LTBP1-proTGFβ1 and mouse LTBP3-proTGFβ1.-   34. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, binds a mouse LTBP1-proTGFβ1    complex with a K_(D) of <50 nM as measured by Bio-Layer    Interferometry (BLI).-   35. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, binds a mouse LTBP3-proTGFβ1    complex with a K_(D) of <50 nM as measured by Bio-Layer    Interferometry (BLI).-   36. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, binds a mouse LTBP1-proTGFβ1    complex with a K_(D) of <10 nM as measured by Bio-Layer    Interferometry (BLI).-   37. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, binds a mouse LTBP3-proTGFβ1    complex with a K_(D) of <10 nM as measured by Bio-Layer    Interferometry (BLI).-   38. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, is isoform-specific in that it    selectively binds and inhibits the activation of TGFβ1 associated    with LTBP1/3.-   39. The antibody, or antigen-binding fragment thereof, according to    embodiment 38, wherein the antibody, or antigen-binding fragment    thereof, does not bind GARP- TGFβ31.-   40. An antibody, or antigen-binding fragment thereof, comprising the    following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTFRSYVMH (SEQ ID NO: 166);

b) CDR-H2 comprising the amino acid sequence VISHEGS(X₁)KYYADSVKG,wherein: X₁ is L or G (SEQ ID NO: 366); and

c) CDR-H3 comprising the amino acid sequence A(X₁)PRIAARRGGFG(X₂),wherein: X₁ is V, R or L; and X₂ is Y, S or T (SEQ ID NO: 367);

d) CDR-L1 comprising the amino acid sequence TRS(X₁)G(X₂)ID(X₃)NYVQ,wherein, X₁ is S or H; X₂ is N, L, S or A; and X₃ is N, D or Y (SEQ IDNO: 368);

e) CDR-L2 comprising the amino acid sequence ED(X₁)(X₂)RPS, wherein: X₁is N, F or A; and X₂ is Q, I or V (SEQ ID NO: 369); and

f) CDR-L3 comprising the amino acid sequence Q(X₁)YD(X₂)(X₃)(X₄)Q(X₅)VV,wherein: X₁ is S or G; X₂ is S, F, Y, D, H or W; X₃ is N, D or S; X₄ isN, A, L, E or T; and X₅ is G, R, A or L (SEQ ID NO: 370).

-   41. The antibody, or antigen-binding fragment thereof, according to    embodiment 40, wherein:

within CDR-H3: X₁ is R or L.

-   42. The antibody, or antigen-binding fragment thereof, according to    embodiment 41, wherein:

within CDR-L3: X₂ is Y.

-   43. The antibody, or antigen-binding fragment thereof, according to    embodiment 42, wherein:

within CDR-L3: X₃ is D; and X₄ is T.

-   44. The antibody, or antigen-binding fragment thereof, according to    embodiment 42, wherein:

within CDR-L3: X₃ is D; X₄ is N; and X₅ is A.

-   45. The antibody, or antigen-binding fragment thereof, according    embodiment 41, wherein:

within CDR-L1: X₁ is S or H; X₂ is N or A; and X₃ is N, D or Y;

within CDR-L2: X₁ is N or F; and X₂ is Q or V; and

within CDR-L3: X₁ is S or G; X₂ is S, Y, D or W; X₃ is D or S; X₄ is N,L or T; and X₅ is G, R, A or L.

-   46. The antibody, or antigen-binding fragment thereof, according to    embodiment 45, wherein:

within CDR-L1: X₁ is S; X₂ is N; and X₃ is N or Y;

within CDR-L2: X₁ is N; and X₂ is Q or V; and

within CDR-L3: X₁ is S or G; X₂ is S, Y or W; X₃ is D; X₄ is N or T; andX₅ is G, R or A.

-   47. The antibody, or antigen-binding fragment thereof, according to    embodiment 46, wherein:

within CDR-L3: X₁ is S; X₂ is S or Y; X₃ is D; X₄ is N or T; and X₅ isG, R or A.

-   48. The antibody, or antigen-binding fragment thereof, according to    embodiment 47, wherein:

within CDR-L3: X₁ is S; X₂ is Y; X₃ is D; X₄ is N or T; and X₅ is G orA.

-   49. The antibody, or antigen-binding fragment thereof, according to    embodiment 48, wherein:

within CDR-L3: X₁ is S; X₂ is Y; X₃ is D; X₄ is T; and X₅ is G.

-   50. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 45-49, wherein:

a) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 166;

b) CDR-H2 comprises the amino acid sequence of SEQ ID NO: 167;

c) CDR-H3 comprises the amino acid sequence of SEQ ID NO: 168;

d) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 169;

e) CDR-L2 comprises the amino acid sequence of SEQ ID NO: 170; and

f) CDR-L3 comprises the amino acid sequence of SEQ ID NO: 171.

-   51. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 40-50, which comprises:

a heavy chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 318; and

a light chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 319.

-   52. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 40-51, which competes or cross-competes with    an antibody having a heavy chain variable region sequence as set    forth in SEQ ID NO: 318 and light chain variable region sequence as    set forth in SEQ ID NO: 319.-   53. An antibody, or antigen-binding fragment thereof, which    selectively binds to human LTBP1-TGFβ1 complex and human LTBP3-TGFβ1    complex and competes or cross-competes with an antibody having a    heavy chain variable region sequence as set forth in SEQ ID NO: 318    and light chain variable region sequence as set forth in SEQ ID NO:    319.-   54. An antibody, or antigen-binding fragment thereof, comprising the    following six CDRs:

g) CDR-H1 comprising the amino acid sequence G(X₁)I(X₂)S(X₃)SYYW(X₄),wherein, optionally: X₁ is S; X₂ is S, H or R; X₃ is S or G; and, X₄ isG, I, N or V (SEQ ID NO: 386);

h) CDR-H2 comprising the amino acid sequence SISYS(X₁)(X₂)TYY, wherein,optionally: X₁ is G or A; and X₂ is S or T (SEQ ID NO: 398);

i) CDR-H3 comprising the amino acid sequence A(X₁)DPSYDS(X₂)AGM(X₃)V,wherein, optionally: X₁ is R, S or G; X₂ is A or I; and X₃ is D or Q(SEQ ID NO: 387);

j) CDR-L1 comprising the amino acid sequence RAS(X₁)(X₂)IS(X₃)YLN,wherein, optionally: X₁ is K or Q; X₂ is V or S; and X₃ is S or Y (SEQID NO: 389);

k) CDR-L2 comprising the amino acid sequence (X₁)AS(X₂)(X₃)QS, wherein,optionally: X₁ is Y, A or S; X₂ is S or N; and X₃ is L or R (SEQ ID NO:390);

l) CDR-L3 comprising the amino acid sequence QQ(X₁)(X₂)D(X₃)P(X₄)T,wherein, optionally: X₁ is S or G; X₂ is F or N; X₃ is W or F; and X₄ isF or L (SEQ ID NO: 391).

-   55. The antibody, or antigen-binding fragment thereof, according to    embodiment 54, wherein:

a) within CDR-H1: X₁ is S; X₂ is S or R; X₃ is 5; and, X₄ is G;

b) within CDR-H2: X₁ is G or A; and X₂ is S or T;

c) within CDR-H3: X₁ is R, S or G; X₂ is A or I; and X₃ is D or Q;

d) within CDR-Ll: X₁ is K or Q; X₂ is V or S; and X₃ is S or Y;

e) within CDR-L2: X₁ is Y, A or S; X₂ is S or N; and X₃ is L or R; and

f) within CDR-L3: X₁ is S or G; X₂ is F or N; X₃ is W or F; and X₄ is For L.

-   56. The antibody, or antigen-binding fragment thereof, according to    embodiment 55, wherein:

a) CDR-H1 comprises the amino acid sequence GSIRSSSYYWG (SEQ ID NO:292);

b) CDR-H2 comprises the amino acid sequence SISYSATTYY (SEQ ID NO: 293);

c) within CDR-H3: X₁ is S or G; X₂ is A or I; and X₃ is D or Q;

d) within CDR-Ll: X₁ is K or Q; X₂ is V or S; and X₃ is S or Y;

e) within CDR-L2: X₁ is Y, A or S; X₂ is S or N; and X₃ is L or R; and

f) within CDR-L3: X₁ is S or G; X₂ is F or N; X₃ is W or F; and X₄ is For L.

-   57. The antibody, or antigen-binding fragment thereof, according to    embodiment 56, wherein:

g) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 292;

h) CDR-H2 comprises the amino acid sequence of SEQ ID NO: 293;

i) CDR-H3 comprises the amino acid sequence of SEQ ID NO: 294;

j) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 295;

k) CDR-L2 comprises the amino acid sequence of SEQ ID NO: 296; and

l) CDR-L3 comprises the amino acid sequence of SEQ ID NO: 297.

-   58. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 54-57, which is an antibody according to any    one of claims 1-5.-   59. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 54-58, which comprises:

a heavy chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 360; and

a light chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 361.

-   60. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 54-59, which competes or cross-competes with    an antibody having a heavy chain variable region sequence as set    forth in SEQ ID NO: 360 and light chain variable region sequence as    set forth in SEQ ID NO: 361.-   61. An antibody, or antigen-binding fragment thereof, which    selectively binds to human LTBP1-TGFβ1 complex and human LTBP3-TGFβ1    complex and competes or cross-competes with an antibody having a    heavy chain variable region sequence as set forth in SEQ ID NO: 360    and light chain variable region sequence as set forth in SEQ ID NO:    361.-   62. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 40-61, which does not show detectable binding    to a human GARP-proTGFβ1 complex, as measured by BLI, under the same    assay conditions as used to measure binding to human LTBP1-proTGFβ1    complex and human LTBP3-TGFβ1 complex.-   63. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 40-62, which binds a human LTBP1-proTGFβ1    complex and/or a human LTBP3-TGFβ1 complex with a K_(D) that is at    least 50 times lower than the K_(D) when binding to a human    GARP-proTGFβ1 complex under the same assay conditions.-   64. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 40-63, which does not show detectable binding    to an LRRC33-proTGFβ1 complex, as measured by BLI, under the same    assay conditions as used to measure binding to human LTBP1-proTGFβ1    complex and human LTBP3-TGFβ1 complex.-   65. The antibody, or antigen-binding fragment thereof, according to    any one of embodiments 40-64, wherein the antibody, or    antigen-binding fragment thereof has a monovalent half-binding-time    t½) of at least 45 minutes for each of hLTBP1-proTGFβ1 and    hLTBP3-proTGFβ1 complexes, as measured by SPR.-   66. The antibody, or antigen-binding fragment thereof, according to    embodiment 65 which has a monovalent t½ of less than 5 minutes for    each of hGARP-proTGFβ1 and hLRRC33-proTGFβ1 complexes, as measured    by SPR.-   67. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, which binds a human    LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K_(D)    of <5 nM as measured by Bio-Layer Interferometry (BLI), optionally    <1 nM.-   68. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, which is cross-reactive with    mouse LTBP1-proTGFβ1.-   69. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, which is cross-reactive with    mouse LTBP3-proTGFβ1.-   70. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, binds a mouse LTBP1-proTGFβ1    complex with a K_(D) of <10 nM as measured by Bio-Layer    Interferometry (BLI).-   71. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof, binds a mouse LTBP3-proTGFβ1    complex with a K_(D) of <10 nM as measured by Bio-Layer    Interferometry (BLI).-   72. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody, or    antigen-binding fragment thereof cross-reacts with human and murine    LTBP1-proTGFβ1 and LTBP3-proTGFβ1 complexes, each with a K_(D) of <5    nM, optionally <1 nM.-   73. The antibody, or antigen-binding fragment thereof, according to    any one of the preceding embodiments, wherein the antibody is an    IgG4 or IgG1 subtype.-   74. The antibody, or antigen-binding fragment thereof, according to    embodiment 73, wherein the antibody is a human IgG4 subtype, wherein    optionally the antibody comprises a backbone substitution of Ser to    Pro that produces an IgG1-like hinge.-   75. A pharmaceutical composition comprising the antibody, or    antigen-binding fragment thereof, according to any one of the    preceding embodiments and a pharmaceutically acceptable excipient.-   76. The pharmaceutical composition according to embodiment 75, which    is prepared for intravenous administration or subcutaneous    administration.-   77. A composition comprising a multi-dose vial containing the    pharmaceutical composition of embodiments 75 or 76.-   78. A composition comprising a single-dose syringe containing the    pharmaceutical composition of embodiments 75 or 76, optionally    wherein the syringe is a disposable syringe.-   79. The composition according to any one of embodiments 75 or 78 for    use in a method for the treatment of a fibrotic condition in a human    subject, wherein the treatment comprises administration of the    composition to the subject in an amount effective to treat the    fibrotic disorder.-   80. The composition for use according to embodiment 79, wherein the    fibrotic disorder is an organ fibrosis.-   81. The composition for use according to embodiment 80, wherein the    organ fibrosis is an advanced organ fibrosis.-   82. The composition for use according to embodiments 80 or 81,    wherein the organ fibrosis is selected from the group consisting of:    kidney fibrosis, liver fibrosis, lung fibrosis, cardiac fibrosis,    pancreatic fibrosis, skin fibrosis, scleroderma, muscle fibrosis,    uterine fibrosis and endometriosis.-   83. The composition for use according to embodiment 82, wherein:

a) the fibrotic disorder comprises chronic inflammation;

b) the subject benefits from immune suppression;

c) the subject has or is at risk of developing an autoimmune disease;

d) the subject is a candidate for an allograft transplant; and/or,

e) the subject has received an allograft transplant.

-   84. The composition for use according to embodiment 79, wherein the    fibrotic disorder comprising chronic inflammation is a muscular    dystrophy, multiple sclerosis (MS), or Cystic Fibrosis (CF).-   85. The composition for use according to embodiment 84, wherein the    muscular dystrophy is Duchenne muscular dystrophy (DMD).-   86. The composition for use according to embodiment 84, wherein the    MS comprises perivascular fibrosis.-   87. The composition for use according to embodiment 82, wherein the    lung fibrosis is idiopathic pulmonary fibrosis (IPF).-   88. The composition for use according to embodiment 82, wherein the    subject has chronic kidney disease (CKD).-   89. The composition for use according to embodiment 82, wherein the    subject has liver fibrosis associated with nonalcoholic    steatohepatitis (NASH) ornonalcoholic fatty liver disease (NAFLD).-   90. The composition for use according to embodiment 89, wherein the    subject has cirrhosis or hepatocellular carcinoma associated with    NASH.-   91. The composition for use according to embodiment 90, wherein the    subject suffers from a metabolic condition (e.g., obesity, type 2    diabetes).-   92. The composition for use according to any one of embodiments    89-91, wherein the the subject is further treated with a myostatin    inhibitor, wherein optionally the myostatin inhibitor is a    myostatin-selective inhibitor, wherein further optionally the    myostatin-selective inhibitor is SRK-015, trevogrumab, or any    variant thereof, or an antibody according to WO 2016/098357.-   93. The composition for use according to any one of embodiments    79-92, wherein the antibody is administered to the subject at a    dosage of between 0.1 and 30 mg/kg.-   94. The composition for use according to embodiment 93, wherein the    antibody is administered twice a week, once a week, once every 2    weeks, once every 3 weeks, once every 4 weeks, once a month, once    every 6 weeks, or every other month.-   95. The composition for use according to embodiment 93, wherein a    therapeutic regimen comprises an initial phase of a therapy and a    subsequent phase of the therapy, wherein the subject receives a    loading dose during the initial phase followed by a maintenance dose    during the subsequent phase.-   96. The composition for use according to embodiment 95, wherein the    loading dose is between 2-30 mg/kg, and the maintenance dose is    between 0.1-20 mg/kg.-   97. The composition for use according to embodiment 95 or 96,    wherein the loading dose is administered to the subject twice a    week,once a week, once every 2 weeks or every 3 weeks.-   98. The composition for use according to any one of embodiments    95-97, wherein the maintenance dose is administered to the subject    once every 2-12 weeks.-   99. The composition for use according to any one of embodiments    95-97, wherein the maintenance dose is administered to the subject    on an as-needed basis.-   100. The composition for use according to any one of embodiments    79-99, wherein the method further comprises testing or confirming    expression of TGFβ1, LTBP1 or LTBP3 in a biological sample collected    from the subject.-   101. A method for making a composition according to any one of    embodiments 75-78, comprising an antibody, or antigen-binding    fragment thereof, that specifically binds a human LTBP1-proTGFβ    complex and/or a human LTBP3-proTGFβ complex, and does not bind a    human GARP-proTGFβ complex; wherein the antibody or fragment    inhibits TGFβ1 but does not inhibit TGFβ2 or TGFβ3, the method    comprising steps of:

i) providing at least one antigen comprising LTBP1-proTGFβ1 and/orLTBP3-proTGFβ31,

ii) selecting a first pool of antibodies or fragments that specificallybind the at least one antigen of step (i) so as to provide specificbinders of LTBP1-proTGFβ1 and/or LTBP3-proTGFβ31;

iii) selecting a second pool of antibodies or fragments that inhibitactivation of TGFβ1, so as to generate specific inhibitors of TGFβ1activation; and

iv) formulating an antibody or fragment that is present in the firstpool of antibodies and the second pool of antibodies into apharmaceutical composition, thereby making the composition comprisingthe antibody or fragment.

-   102. The method of embodiment 101, wherein the method further    comprises a step of:

removing from the first pool of antibodies, or fragments, any antibodiesor fragments that bind GARP-proTGFβ1, LRRC33-proTGFβ1, mature TGFβ1,GARP-proTGFβ2, LRRC33-proTGFβ2, mature TGFβ2, GARP-proTGFβ3,LRRC33-proTGFβ3, mature TGFβ3, or any combinations thereof.

-   103. The method of embodiment 101 or 102, wherein the method further    comprises a step of:

determining or confirming isoform-specificity of the antibodies orfragments selected in steps (ii) and/or (iii).

-   104. The method according to any one of embodiments 101-103, wherein    the method further comprises a step of:

selecting antibodies or fragments that are cross-reactive to human androdent antigens.

-   105. The method according to any one of embodiments 101-104, wherein    the method further comprises a step of:

generating a fully human or humanized antibody or fragment, of theantibody or fragment that is presented in the first pool of antibodiesand the second pool of antibodies.

-   106. The method according to any one of embodiments 101-105, wherein    the method further comprises a step of:

subjecting the antibody or fragment that is present in the first pool ofantibodies and the second pool of antibodies to affinity maturationand/or optimization, so as to provide an affinity matured and/oroptimized antibody or fragment.

-   107. The method according to any one of embodiments 101-106, wherein    the affinity maturation and/or optimization comprises a step of    subjecting the antibody, or antigen-binding fragment thereof, to    light chain shuffling.-   108. The method according to any one of embodiments 101-107, wherein    the affinity maturation and/or optimization comprises the step of    subjecting the antibody, or antigen-binding fragment thereof, to CDR    H1/H2 diversification.-   109. The method according to any one of embodiments 101-108, wherein    the affinity maturation and/or optimization comprises the step of    subjecting the antibody, or antigen-binding fragment thereof, to    CDR-H3 mutagenesis.-   110. The method according to any one of embodiments 101-109, wherein    the affinity maturation and/or optimization comprises the step of    subjecting antibody, or antigen-binding fragment thereof, to light    chain CDR mutagenesis.-   111. The method according to any one of embodiments 101-110, wherein    the affinity maturation and/or optimization comprises the step of    subjecting the antibody, or antigen-binding fragment thereof, to    light chain CDR L1/L2 diversification.-   112. The method according to any one of embodiments 101-111, wherein    the method further comprises a step of determining affinity of the    antibodies, or antigen-binding fragments thereof, to human    LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1.-   113. The method according to embodiments 101-112, wherein the method    further comprises a step of removing from the first and/or second    pools of antibodies, or antigen-binding fragments thereof, any    antibodies, or antigen-binding fragments thereof, that bind to human    LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K_(D) of >100 nM,    as measured by Bio-Layer Interferometry (BLI).-   114. The method according to embodiment 101-113, wherein the method    further comprises a step of removing from the first and/or second    pools of antibodies, or antigen-binding fragments thereof, any    antibodies, or antigen-binding fragments thereof, that bind to human    LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K_(D) of >50 nM,    as measured by Bio-Layer Interferometry (BLI).-   115. The method according to embodiment 101-114, wherein the method    further comprises a step of removing from the first and/or second    pools of antibodies, or antigen-binding fragments thereof, any    antibodies, or antigen-binding fragments thereof, that bind to human    LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K_(D) of >25 nM,    as measured by Bio-Layer Interferometry (BLI).-   116. The method according to embodiment 101-115, wherein the method    further comprises a step of removing from the first and/or second    pools of antibodies, or antigen-binding fragments thereof, any    antibodies, or antigen-binding fragments thereof, that bind to human    LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K_(D) of >10 nM,    as measured by Bio-Layer Interferometry (BLI).-   117. The method according to any one of embodiments 101-116, wherein    the method further comprises a step of determining affinity of the    antibodies, or antigen-binding fragments thereof, from the first    and/or second pools of antibodies to mouse LTBP1-proTGFβ1 and/or    mouse LTBP3-proTGFβ31.-   118. The method according to embodiment 117, wherein the method    further comprises a step of removing from the first and/or second    pools of antibodies, or antigen-binding fragments thereof, any    antibodies, or antigen-binding fragments thereof, that bind to mouse    LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K_(D) of >100 nM,    as measured by Bio-Layer Interferometry (BLI).-   119. The method according to embodiment 117, wherein the method    further comprises a step of removing from the first and/or second    pools of antibodies, or antigen-binding fragments thereof, any    antibodies, or antigen-binding fragments thereof, that bind to mouse    LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K_(D) of >50 nM,    as measured by Bio-Layer Interferometry (BLI).-   120. The method according to embodiment 117, wherein the method    further comprises a step of removing from the first and/or second    pools of antibodies, or antigen-binding fragments thereof, any    antibodies, or antigen-binding fragments thereof, that bind to mouse    LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K_(D) of >25 nM,    as measured by Bio-Layer Interferometry (BLI).-   121. The method according to embodiment 117, wherein the method    further comprises a step of removing from the first and/or second    pools of antibodies, or antigen-binding fragments thereof, any    antibodies, or antigen-binding fragments thereof, that bind to mouse    LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K_(D) of >10 nM,    as measured by Bio-Layer Interferometry (BLI).-   122. The method according to any one of embodiments 101-121, wherein    the method further comprises a step of removing from the first    and/or second pool of antibodies, or antigen-binding fragments    thereof, any antibodies, or antigen-binding fragments thereof, that    do not bind mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1.-   123. The method according to any one of embodiments 101-122, wherein    the method further comprises a step of determining the IC₅₀ of the    antibodies, or antigen-binding fragments thereof, from the first    and/or second pools of antibodies, or antigen-binding fragments    thereof, as measured by a suitable functional in vitro cell-based    assay.-   124. The method according to embodiments 123, wherein the suitable    functional in vitro cell-based assay is a caga assay.-   125. The method according to embodiments 123 or 124, wherein the    method comprises the step of removing antibodies, or antigen-binding    fragments thereof, from the first and/or second pools of antibodies,    or antigen-binding fragments thereof, that have an IC₅₀ of >100 nM.-   126. The method according to embodiments 123 or 124, wherein the    method comprises the step of removing antibodies, or antigen-binding    fragments thereof, from the first and/or second pools of antibodies,    or antigen-binding fragments thereof, that have an IC₅₀ of >50 nM.-   127. The method according to embodiments 123 or 124, wherein the    method comprises the step of removing antibodies, or antigen-binding    fragments thereof, from the first and/or second pools of antibodies,    or antigen-binding fragments thereof, that have an IC₅₀ of >25 nM.-   128. The method according to embodiments 123 or 124, wherein the    method comprises the step of removing antibodies, or antigen-binding    fragments thereof, from the first and/or second pools of antibodies,    or antigen-binding fragments thereof, that have an IC₅₀ of >10 nM.-   129. The method according to embodiments 123 or 124, wherein the    method comprises the step of removing antibodies, or antigen-binding    fragments thereof, from the first and/or second pools of antibodies,    or antigen-binding fragments thereof, that have an IC₅₀ of >5 nM.-   130. The method according to any one of embodiments 124-129, wherein    the caga assay is an endogenous LTBP caga assay.-   131. The method according to any one of embodiments 124-129, wherein    the caga assay is a human LTBP overexpression caga assay.-   132. The method according to any one of embodiments 124-129, wherein    the caga assay is a murine LTBP overexpression caga assay.-   133. A method for manufacturing a pharmaceutical composition    comprising an antibody, or antigen-binding fragment thereof, that    selectively binds a human LTBP1-proTGFβ complex and/or a human    LTBP3-proTGFβ complex, the method comprosing the steps of:

selecting an antibody or antigen-binidng fragment thereof thatpreferentailly inhibits matrix-associated TGFβ1 over immunecell-associated TGFβ1;

producing the antibody in a cell culture comprising cells expressing theantibody, wherein the cell culture has a volume of 250L or greater,

optionally further comprising the step of purifying the antibody fromthe cell culture; and

further optionally comprising the step of formulating the purisedantibody into a pharmaceutical composition.

-   134. The method according to 133, wherein the pharmaceutical    composition is formulated for subcutaneous administration.-   135. The method according to 133, wherein the selection step further    comprises selecting an antibody or antigen-binding fragment that has    t½ of 45 minutes or longer for each of human LTBP1-proTGFβ and human    LTBP3-proTGFβ complexes and optionally t½ of 5 minutes or less for    human GARP-proTGFβ complex, as measured by SPR.-   136. A method for making an antibody, or an antigen-binding fragment    thereof, the method comprising steps of:

i) providing an antibody or a fragment that comprises at least threeheavy chain CDR sequences of (CDR-H1) SEQ ID NO:94, (CDR-H2) SEQ IDNO:95, and (CDR-H3) SEQ ID NO:96; and

ii) subjecting the antibody or fragment of step (i) to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody or fragment.

-   137. The method according to embodiment 136, wherein the antibody,    or antigen-binding fragment thereof, further comprises light chain    CDR sequences of (CDR-L1) SEQ ID NO:97, (CDR-L2) SEQ ID NO:98, and    (CDR-L3) SEQ ID NO:99.-   138. The method according to embodiments 136 or 137, wherein the    antibody or fragment comprises a variable heavy chain region having    an amino acid sequence as set forth in SEQ ID NO: 88.-   139. The method according to any one of embodiments 136-138, wherein    the antibody or fragment comprises a variable light chain region    having an amino acid sequence as set forth in SEQ ID NO: 89.-   140. A method for making an antibody, or an antigen-binding fragment    thereof, the method comprising steps of:

i) providing an antibody or a fragment that comprises at least three CDRsequences of (CDR-H1) SEQ ID NO:100, (CDR-H2) SEQ ID NO:101, and(CDR-H3) SEQ ID NO:102; and

ii) subjecting the antibody or fragment of step (i) to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody or fragment.

-   141. The method of embodiment 140, wherein the antibody, or    antigen-binding fragment thereof, further comprises light chain CDR    sequences of (CDR-L1) SEQ ID NO:103, (CDR-L2) SEQ ID NO:104, and    (CDR-L3) SEQ ID NO:105.-   142. The method according to embodiments 140 or 141, wherein the    antibody or fragment comprises a variable heavy chain region having    an amino acid sequence as set forth in SEQ ID NO: 106.-   143. The method according to any one of embodiments 140-142, wherein    the antibody or fragment comprises a variable light chain region    having an amino acid sequence as set forth in SEQ ID NO: 107.-   144. A method for making an antibody, or an antigen-binding fragment    thereof, the method comprising steps of:

i) providing an antibody or a fragment that comprises at least threeheavy chain CDR sequences of (CDR-H1) SEQ ID NO:108, (CDR-H2) SEQ IDNO:109, and (CDR-H3) SEQ ID NO:110 and

ii) subjecting the antibody or fragment of step (i) to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody or fragment.

-   145. The method according to embodiment 144, wherein the antibody,    or antigen-binding fragment thereof, further comprises light chain    CDR sequences of (CDR-L1) SEQ ID NO:111, (CDR-L2) SEQ ID NO:112, and    (CDR-L3) SEQ ID NO:113.-   146. The method according to embodiments 144or 145, wherein the    antibody or fragment comprises a variable heavy chain region having    an amino acid sequence as set forth in SEQ ID NO: 114.-   147. The method according to any one of embodiments 144-146, wherein    the antibody or fragment comprises a variable light chain region    having an amino acid sequence as set forth in SEQ ID NO: 115.-   148. A method for making an antibody, or an antigen-binding fragment    thereof, the method comprising steps of:

i) providing an antibody or a fragment that comprises at least threeheavy chain CDR sequences of (CDR-H1) SEQ ID NO:116, (CDR-H2) SEQ IDNO:117, and (CDR-H3) SEQ ID NO:118 and

ii) subjecting the antibody or fragment of step (i) to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody or fragment.

149. The method according to embodiment 148, wherein the antibody, orantigen-binding fragment thereof, further comprises light chain CDRsequences of (CDR-L1) SEQ ID NO:119, (CDR-L2) SEQ ID NO:120, and(CDR-L3) SEQ ID NO:121.

-   150. The method according to embodiments 148 or 149, wherein the    antibody or fragment comprises a variable heavy chain region having    an amino acid sequence as set forth in SEQ ID NO: 122.-   151. The method according to any one of embodiments 148-150, wherein    the antibody or fragment comprises a variable light chain region    having an amino acid sequence as set forth in SEQ ID NO: 123.

152. The method according to any one of embodiments 136-151, whereinstep (ii) comprises light-chain shuffling.

-   153. The method according to any one of embodiments 136-152, wherein    step (ii) comprises CDR-H1/H2 diversification.-   154. The method according to any one of embodiments 136-153, wherein    step (ii) comprises CDR-L1/L2 diversification.-   155. The method according to any one of embodiments 136-154, wherein    step (ii) comprises mutagenesis within any one of the CDRs, variable    regions, and/or constant regions.-   156. The method according to embodiment 155, wherein the mutagenesis    is within a CDR.-   157. The method according to any one of embodiments 155 and 156,    wherein the mutagenesis is within a CDR-H3.-   158. The method according to any one of embodiments 155-157, wherein    the mutagenesis is within a CDR-L1.-   159. The method according to any one of embodiments 155-158, wherein    the mutagenesis is within a CDR-L2.-   160. The method according to any one of embodiments 155-159, wherein    the mutagenesis is within a CDR-L3.-   161. The method according to embodiment 155, wherein the mutagenesis    is within a variable region.-   162. The method according to embodiment 155, wherein the mutagenesis    is within a constant region.-   163. The method of any one of embodiments 136-162, wherein the    method further comprises a step of:

selecting affinity matured and/or optimized antibodies, orantigen-binding fragments thereof, that do not bind GARP-proTGFβ1,LRRC33-proTGFβ1, mature TGFβ1, GARP-proTGFβ2, LRRC33-proTGFβ2, matureTGFβ2, GARP-proTGFβ3, LRRC33-proTGFβ3, mature TGFβ3, or any combinationsthereof.

-   164. The method of any one of embodiments 136-163, wherein the    method further comprises a step of:

determining or confirming isoform-specificity of the affinity maturedand/or optimized antibodies or fragments.

-   165. The method according to any one of embodiments 136-164, wherein    the method further comprises a step of:

selecting antibodies or fragments that are cross-reactive to human androdent antigens.

-   166. The method according to any one of embodiments 136-165, wherein    the method further comprises a step of:

generating a fully human or humanized antibody or fragment, of theaffinity matured and/or optimized antibody or fragment.

-   167. The method according to any one of embodiments 136-166, wherein    the method further comprises a step of:

determining affinity of the affinity matured and/or optimizedantibodies, or fragments, to human LTBP1-proTGFβ1 and/or humanLTBP3-proTGFβ1.

-   168. The method according to embodiments 136-167, wherein the method    further comprises a step of: selecting affinity matured and/or    optimized antibodies, or fragments, that bind to human    LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K_(D) of <100 nM,    as measured by Bio-Layer Interferometry (BLI).-   169. The method according to embodiment 136-168, wherein the method    further comprises a step of: selecting affinity matured and/or    optimized antibodies, or fragments, that bind to human    LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K_(D) of <50 nM,    as measured by Bio-Layer Interferometry (BLI).-   170. The method according to embodiment 136-169, wherein the method    further comprises a step of: selecting affinity matured and/or    optimized antibodies, or fragments, that bind to human    LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K_(D) of <25 nM,    as measured by Bio-Layer Interferometry (BLI).-   171. The method according to embodiment 136-170, wherein the method    further comprises a step of: selecting affinity matured and/or    optimized antibodies, or fragments, that bind to human    LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K_(D) of <10 nM,    as measured by Bio-Layer Interferometry (BLI).-   172. The method according to any one of embodiments 136-171, wherein    the method further comprises a step of:

determining affinity of the affinity matured and/or optimized antibodiesor fragments, to mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1.

-   173. The method according to embodiment 136-172, wherein the method    further comprises a step of: selecting affinity matured and/or    optimized antibodies, or fragments, that bind to mouse    LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K_(D) of <100 nM,    as measured by Bio-Layer Interferometry (BLI).-   174. The method according to embodiment 136-173, wherein the method    further comprises a step of: selecting affinity matured and/or    optimized antibodies, or fragments, that bind to mouse    LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K_(D) of <50 nM,    as measured by Bio-Layer Interferometry (BLI).-   175. The method according to embodiment 136-174, wherein the method    further comprises a step of: selecting affinity matured and/or    optimized antibodies, or fragments, that bind to mouse    LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K_(D) of <25 nM,    as measured by Bio-Layer Interferometry (BLI).-   176. The method according to embodiment 136-175, wherein the method    further comprises a step of: selecting affinity matured and/or    optimized antibodies, or fragments, that bind to mouse    LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K_(D) of <10 nM,    as measured by Bio-Layer Interferometry (BLI).-   177. The method according to any one of embodiments 136-176, wherein    the method further comprises a step of:

selecting affinity matured and/or optimized antibodies, or fragments,that do not bind mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1.

-   178. The method according to any one of embodiments 136-177, wherein    the method further comprises a step of:

determining the IC₅₀ of the affinity matured and/or optimizedantibodies, or fragments, as measured by a suitable functional in vitrocell-based assay.

-   179. The method according to embodiments 178, wherein the suitable    functional in vitro cell-based assay is a caga assay.

180. The method according to any one of embodiments 136-179, wherein themethod comprises the step of:

selecting affinity matured and/or optimized antibodies, or fragments,that have an IC₅₀ of <100 nM.

-   181. The method according to any one of embodiments 136-179, wherein    the method comprises the step of:

selecting affinity matured and/or optimized antibodies, or fragments,that have an IC₅₀ of <50 nM.

-   182. The method according to embodiments 136-179, wherein the method    comprises the step of:

selecting affinity matured and/or optimized antibodies, or fragments,that have an IC₅₀ of <25 nM.

-   183. The method according to embodiments 136-179, wherein the method    comprises the step of:

selecting affinity matured and/or optimized antibodies, or fragments,that have an IC₅₀ of <10 nM.

-   184. The method according to embodiments 136-179, wherein the method    comprises the step of:

selecting affinity matured and/or optimized antibodies, or fragments,that have an IC₅₀ of <5 nM.

-   185. The method according to any one of embodiments 179-184, wherein    the caga assay is an endogenous LTBP caga assay.-   186. The method according to any one of embodiments 179-184, wherein    the caga assay is a human LTBP overexpression caga assay.-   187. The method according to any one of embodiments 179-184, wherein    the caga assay is a murine LTBP overexpression caga assay.-   188. The antibody or the fragment, use thereof, or related methods    thereof, according to any one of the preceding embodiments,

a) which comprises the following variable heavy and/or variable lightchain sequences, or a variant thereof:

-   -   i) the variable heavy chain sequences        QVQLVESGGGVVQPGRSLRLSCAASGFTFRSYVMHWVRQAPGKGLEWVAVISHEGSLKYY        ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVPRIAARRGGFGYWGQGTLVTVSS        (SEQ ID NO: 114) or        QVQLVESGGGVVQPGRSLRLSCAASGFTFRSYVMHWVRQAPGKGLEWVAVISHEGSLKYY AD        SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPRIAARRGGFGYWGQGTLVTVS S        (SEQ ID NO: 318), wherein optionally the variant is at least 85%        identical, 90% identical, or 95% identical to the corresponding        variable heavy chain sequence.    -   ii) the variable light chain sequences

(SEQ ID NO: 89) NFMLTQPHSVSESPGKTVTISCTRSSGNIDNNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSDNQG VVFGGGTKLTVL or(SEQ ID NO: 89) NFMLTQPHSVSESPGKTVTISCTRSSGNIDNNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSDNQG VVFGGGTKLTVL,wherein optionally the variant is at least 85% identical, 90% identical,or 95% identical to the corresponding variable light chain sequence;

b) which has the following variable heavy chain CDR sequences, orvariant thereof:

-   -   i) CDR-H1 of FTFRSYVMH (SEQ ID NO: 166) optionally comprising        one or more amino acid changes;    -   ii) CDR-H2 of VISHEGSLKYYADSVKG (SEQ ID NO: 167) optionally        comprising one or more amino acid changes; and/or;    -   iii) CDR-H3 of AVPRIAARRGGFGY (SEQ ID NO: 110) or ARPRIAARRGGFGY        (SEQ ID NO: 168) optionally comprising one or more amino acid        changes; and/or;

c) which has the following variable light chain CDR sequences, orvariant thereof:

-   -   i) CDR1-L1 of TRSSGNIDNNYVQ (SEQ ID NO: 169) optionally        comprising one or more amino acid changes;    -   ii) CDR-L2 of EDNQRPS (SEQ ID NO: 170) optionally comprising one        or more amino acid changes; and/or;    -   iii) CDR-L3 of QSYDSDNQGVV (SEQ ID NO: 113) optionally        comprising one or more amino acid changes.

-   A1. An isolated antibody that specifically binds a human    LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex, and does    not bind a human GARP-proTGFβ complex;

wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or matureTGFβ3;

wherein the antibody is a fully human or humanized antibody, orantigen-binding fragment thereof,

wherein the antibody comprises at least three CDRs selected from thefollowing, optionally comprising up to 3 amino acid changes for each ofthe CDRs:

CDR-Hl: SEQ ID NO:100 ;

CDR-H2: SEQ ID NO:101;

CDR-H3: SEQ ID NO:102;

CDR-Ll: SEQ ID NO:103;

CDR-L2: SEQ ID NO:104; and,

CDR-L3: SEQ ID NO:105.

-   A1.1. An isolated antibody that specifically binds a human    LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex, and does    not bind a human GARP-proTGFβ complex;

wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or matureTGFβ3;

wherein the antibody is a fully human or humanized antibody, orantigen-binding fragment thereof,

wherein the antibody comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:100 (SEQ ID NO: 100 comprising these substitutionsis disclosed as SEQ ID NO: 400), with the proviso that:

-   -   i. the serine residue at position 4 of SEQ ID NO:100 may be        substituted with a histidine;    -   ii. the serine residue at position 7 of SEQ ID NO:100 may be        substituted with an alanine or glycine; and/or,    -   iii. the glycine residue at position 11 of SEQ ID NO:100 may be        substituted with a threonine, serine, histidine, leucine,        isoleucine, asparagine, valine, or alanine;

b) CDR-H2: SEQ ID NO:101 (SEQ ID NO: 101 comprising these substitutionsis disclosed as SEQ ID NO: 401), with the proviso that:

-   -   i. the serine residue at position 3 of SEQ ID NO:101 may be        substituted with an alanine;    -   ii. the glycine residue at position 6 of SEQ ID NO:101 may be        substituted with an alanine or serine; and/or,    -   iii. the serine residue at position 7 of SEQ ID NO:101 may be        substituted with a threonine;

c) CDR-H3: SEQ ID NO:102, optionally comprising up to three amino acidchanges;

d) CDR-L1: SEQ ID NO:103, optionally comprising up to three amino acidchanges;

e) CDR-L2: SEQ ID NO:104, optionally comprising up to three amino acidchanges; and,

f) CDR-L3: SEQ ID NO:105, optionally comprising up to three amino acidchanges.

-   A2. An isolated antibody that specifically binds a human    LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex, and does    not bind a human GARP-proTGFβ complex;

wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or matureTGFβ3;

wherein the antibody is a fully human or humanized antibody, orantigen-binding fragment thereof;

wherein the antibody comprises a heavy chain variable region having anamino acid sequence that is at least 90% identical to SEQ ID NO: SEQ IDNO: 106; and/or,

wherein the antibody comprises a variable light chain variable regionhaving an amino acid sequence that is at least 90% identical to SEQ IDNO: 107.

-   A3. The antibody according to embodiment A2, wherein the antibody    comprises:

a heavy chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 106; and

a light chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 107.

-   A3.1 An isolated antibody that specifically binds a human    LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex, and does    not bind a human GARP-proTGFβ complex;

wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or matureTGFβ33;

wherein the antibody is a fully human or humanized antibody, orantigen-binding fragment thereof,

wherein the antibody comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:94 (SEQ ID NO: 94 comprising these substitutions isdisclosed as SEQ ID NO: 399), with the proviso that:

-   -   i. the threonine residue at position 2 of SEQ ID NO:94 may be        substituted with an alanine;    -   ii. the asparagine residue at position 4 of SEQ ID NO:94 may be        substituted with an alanine, tyrosine, aspartate, serine,        arginine, or histidine;    -   iii. the asparagine residue at position 5 of SEQ ID NO:94 may be        substituted with a glutamine, serine, glycine, lysine,        glutamate, arginine, or histidine;    -   iv. the tyrosine residue at position 6 of SEQ ID NO:94 may be        substituted with a arginine;    -   v. the proline residue at position 7 of SEQ ID NO:94 may be        substituted with a glycine, alanine, leucine, serine,        asparagine, valine, aspartate, or glutamine;    -   vi. the isoleucine residue at position 8 of SEQ ID NO:94 may be        substituted with a methionine or leucine; and/or,    -   vii. the histidine residue at position 9 of SEQ ID NO:94 may be        substituted with a phenylalanine, tyrosine, asparagine, or        serine;

b) CDR-H2: SEQ ID NO:95, optionally comprising up to six amino acidchanges;

c) CDR-H3: SEQ ID NO:96, optionally comprising up to three amino acidchanges;

d) CDR-L1: SEQ ID NO:97, optionally comprising up to three amino acidchanges;

e) CDR-L2: SEQ ID NO:98, optionally comprising up to three amino acidchanges; and,

f) CDR-L3: SEQ ID NO:99, optionally comprising up to three amino acidchanges.

-   A3.2. An isolated antibody that specifically binds a human    LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex, and does    not bind a human GARP-proTGFβ complex;

wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or matureTGFβ3;

wherein the antibody is a fully human or humanized antibody, orantigen-binding fragment thereof;

wherein the antibody comprises a heavy chain variable region having anamino acid sequence that is at least 90% identical to SEQ ID NO: SEQ IDNO: 88; and/or, wherein the antibody comprises a variable light chainvariable region having an amino acid sequence that is at least 90%identical to SEQ ID NO: 89.

-   A3.3. The antibody according to embodiment A3.2, wherein the    antibody comprises:

a heavy chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 88; and

a light chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 89.

-   A3.4. An isolated antibody that specifically binds a human    LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex, and does    not bind a human GARP-proTGFβ complex;

wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or matureTGFβ3;

wherein the antibody is a fully human or humanized antibody, orantigen-binding fragment thereof,

wherein the antibody comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:1, with the proviso that:

-   -   i. the threonine residue at position 4 of SEQ ID NO:1 may be        substituted with a histidine, lysine, phenylalanine, or glycine;    -   ii. the serine residue at position 5 of SEQ ID NO:1 may be        substituted with an leucine; and/or,    -   iii. the serine residue at position 9 of SEQ ID NO:1 may be        substituted with an alanine;

b) CDR-H2: SEQ ID NO:2, with the proviso that:

-   -   i. the serine residue at position 3 of SEQ ID NO:2 may be        substituted with an aspartate or asparagine;    -   ii. the tyrosine residue at position 5 of SEQ ID NO:2 may be        substituted with a histidine;    -   iii. the asparagine residue at position 6 of SEQ ID NO:2 may be        substituted with a serine;    -   iv. the asparagine residue at position 8 of SEQ ID NO:2 may be        substituted with a phenylalanine, leucine, alanine, tyrosine,        aspartate, or serine; and/or,    -   v. the asparagine residue at position 10 of SEQ ID NO:2 may be        substituted with a aspartate or alanine;

c) CDR-H3: SEQ ID NO:3, optionally comprising up to three amino acidchanges;

d) CDR-L1: SEQ ID NO:4, optionally comprising up to three amino acidchanges;

e) CDR-L2: SEQ ID NO:5, optionally comprising up to three amino acidchanges; and,

f) CDR-L3: SEQ ID NO:6, optionally comprising up to three amino acidchanges.

A3.5. An isolated antibody that specifically binds a human LTBP1-proTGFβcomplex and/or a human LTBP3-proTGFβ complex, and does not bind a humanGARP-proTGFβ complex;

wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or matureTGFβ3;

wherein the antibody is a fully human or humanized antibody, orantigen-binding fragment thereof;

wherein the antibody comprises a heavy chain variable region having anamino acid sequence that is at least 90% identical to SEQ ID NO: SEQ IDNO: 7; and/or, wherein the antibody comprises a variable light chainvariable region having an amino acid sequence that is at least 90%identical to SEQ ID NO: 8.

-   A3.6. The antibody according to embodiment A3.5, wherein the    antibody comprises:

a heavy chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 7; and

a light chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO: 8.

-   A4. The antibody according to any one of embodiments Al-A3.6,    wherein the antibody does not compete with any one of the antibodies    SR-Abl, SR-Ab2 or SR-Abl0 for binding to a human LTBP1-proTGFβ1    complex.-   A5. The antibody according to any one of embodiments Al-A4, wherein    the antibody is an IgG4 or IgG1 subtype.-   A5.1 The antibody according to any one of embodiments Al-A5, wherein    the antibody is specific for human LTBP1-TGFβ1 complex.-   A5.2 The antibody according to any one of embodiments Al-A5.1,    wherein the antibody is specific for human LTBP3-TGFβ1 complex.-   A5.3 The antibody according to any one of embodiments Al-A5.2,    wherein the antibody is specific for human LTBP1-TGFβ1 complex and    human LTBP3-TGFβ1 complex.-   A5.4 The antibody according to any one of embodiments Al-A5.3,    wherein the antibody does not bind a human GARP-TGFβ1 complex or    GARP-TGFβ3 complex.-   A6. A pharmaceutical composition comprising the antibody of any one    of embodiments Al-A5.4 and a pharmaceutically acceptable excipient.-   A7. The pharmaceutical composition according to embodiment A6, which    is prepared for intravenous administration or subcutaneous    administration.-   A8. A composition comprising a multi-dose vial containing the    pharmaceutical composition of embodiment A6 or A7.-   A9. A composition comprising a single-dose syringe containing the    pharmaceutical composition of embodiment A6 or A7, optionally    wherein the syringe is a disposable syringe.-   A10. The composition of any one of embodiments A6-A9 for use in a    method for the treatment of a fibrotic condition in a human subject,    wherein the treatment comprises administration of the composition to    the subject in an amount effective to treat the fibrotic disorder.-   A11. The composition for use according to embodiment A10, wherein    the fibrotic disorder is an organ fibrosis.-   A12. The composition for use according to embodiment All, wherein    the organ fibrosis is an advanced organ fibrosis.-   A13. The composition for use according to embodiment A10 or All,    wherein the organ fibrosis is selected from the group consisting of:    kidney fibrosis, liver fibrosis, lung fibrosis, cardiac fibrosis,    pancreatic fibrosis, skin fibrosis, scleroderma, muscle fibrosis,    uterine fibrosis and endometriosis.-   A14. The composition for use according to embodiment A10, wherein:

a) the fibrotic disorder comprises chronic inflammation;

b) the subject benefits from immune suppression;

c) the subject has or is at risk of developing an autoimmune disease;

d) the subject is a candidate for an allograft transplant; and/or,

e) the subject has received an allograft transplant.

-   A15. The composition for use according to embodiment A10, wherein    the fibrotic disorder comprising chronic inflammation is a muscular    dystrophy, multiple sclerosis (MS), or Cystic Fibrosis (CF).-   A16. The composition for use according to embodiment A15, wherein    the muscular dystrophy is Duchenne muscular dystrophy (DMD).-   A17. The composition for use according to embodiment A15, wherein    the MS comprises perivascular fibrosis.-   A18. The composition for use according to embodiment A13, wherein    the lung fibrosis is idiopathic pulmonary fibrosis (IPF).-   A19. The composition for use according to embodiment A13, wherein    the subject has chronic kidney disease (CKD).-   A20. The composition for use according to embodiment A13, wherein    the subject has nonalcoholic steatohepatitis (NASH).-   A21. The composition for use according to any one of embodiments    A10-A20, wherein the antibody is administered to the subject at a    dosage of between 0.1 and 30 mg/kg.-   A22. The composition for use according to embodiment A21, wherein    the antibody is administered twice a week, once a week, once every 2    weeks, once every 3 weeks, once a month, or every other month.

A23. The composition for use according to embodiment A21, wherein atherapeutic regimen comprises an initial phase of a therapy and asubsequent phase of the therapy,

wherein the subject receives a loading dose during the initial phasefollowed by a maintenance dose during the subsequent phase.

-   A24. The composition for use according to embodiment A21, wherein    the loading dose is between 2-30 mg/kg, and the maintenance dose is    between 0.1-20 mg/kg.-   A25. The composition for use according to embodiment A21, wherein    the loading dose is administered to the subject twice a week or once    a week.-   A26. The composition for use according to embodiment A21, wherein    the maintenance dose is administered to the subject once every 2-8    weeks.-   A27. The composition for use according to any one of embodiments    A10-A26, wherein the method further comprises testing or confirming    expression of TGFβ1, LTBP1 or LTBP3 in a biological sample collected    from the subject.-   A28. A method for making a composition of any one of embodiments    A6-A9, comprising an antibody, or antigen-binding fragment thereof,    that specifically binds a human LTBP1-proTGFβ complex and/or a human    LTBP3-proTGFβ complex, and does not bind a human GARP-proTGFβ    complex; wherein the antibody or fragment inhibits TGFβ1 but does    not inhibit TGFβ2 or TGFβ3, the method comprising steps of:

i) providing at least one antigen comprising LTBP1-proTGFβ1 and/orLTBP3-proTGFβ31,

ii) selecting a first pool of antibodies or fragments that specificallybind the at least one antigen of step (i) so as to provide specificbinders of LTBP1-proTGFβ1 and/or LTBP3-proTGFβ31;

iii) selecting a second pool of antibodies or fragments that inhibitactivation of TGFβ1, so as to generate specific inhibitors of TGFβ1activation; and

iv) formulating an antibody or fragment that is present in the firstpool of antibodies and the second pool of antibodies into apharmaceutical composition, thereby making the composition comprisingthe antibody or fragment.

-   A29. The method of embodiment A28, wherein the method further    comprises a step of:

removing from the first pool of antibodies, or fragments, any antibodiesor fragments that bind GARP-proTGFβ1, LRRC33-proTGFβ1, mature TGFβ1,GARP-proTGFβ2, LRRC33-proTGFβ2, mature TGFβ2, GARP-proTGFβ3,LRRC33-proTGFβ3, mature TGFβ3, or any combinations thereof.

-   A30. The method of embodiment A28 or A29, wherein the method further    comprises a step of:

determining or confirming isoform-specificity of the antibodies orfragments selected in steps (ii) and/or (iii).

-   A31. The method of any one of embodiments A28-A30, wherein the    method further comprises a step of:

selecting antibodies or fragments that are cross-reactive to human androdent antigens.

-   A32. The method of any one of embodiments A28-A31, wherein the    method further comprises a step of:

generating a fully human or humanized antibody or fragment, of theantibody or fragment that is presented in the first pool of antibodiesand the second pool of antibodies.

-   A33. The method of any one of embodiments A28-A32, wherein the    method further comprises a step of:

subjecting the antibody or fragment that is present in the first pool ofantibodies and the second pool of antibodies to affinity maturationand/or optimization, so as to provide an affinity matured and/oroptimized antibody or fragment.

-   A34. A method for making an antibody, or an antigen-binding fragment    thereof, the method comprising steps of:

i) providing an antibody or a fragment that comprises at least three CDRsequences of (CDR-H1) SEQ ID NO:1, (CDR-H2) SEQ ID NO:2, and (CDR-H3)SEQ ID NO:3; and

ii) subjecting the antibody or fragment of step (i) to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody or fragment.

-   A35. A method for making an antibody, or an antigen-binding fragment    thereof, the method comprising steps of:

i) providing an antibody or a fragment that comprises at least three CDRsequences of (CDR-H1) SEQ ID NO:94, (CDR-H2) SEQ ID NO:95, and (CDR-H3)SEQ ID NO:96; and

ii) subjecting the antibody or fragment of step (i) to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody or fragment.

-   A36. A method for making an antibody, or an antigen-binding fragment    thereof, the method comprising steps of:

i) providing an antibody or a fragment that comprises at least three CDRsequences of (CDR-H1) SEQ ID NO:100, (CDR-H2) SEQ ID NO:101, and(CDR-H3) SEQ ID NO:102; and

ii) subjecting the antibody or fragment of step (i) to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody or fragment.

-   A37. A method for making an antibody, or an antigen-binding fragment    thereof, the method comprising steps of:

i) providing an antibody or a fragment that comprises a heavy chainvariable region having an amino acid sequence set forth in SEQ ID NO: 7;and

ii) subjecting the antibody or fragment of step (i) to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody, or fragment.

-   A38. A method for making antibody, or an antigen-binding fragment    thereof, the method comprising steps of:

i) providing an antibody or a fragment that comprises a heavy chainvariable region having an amino acid sequence set forth in SEQ ID NO:88; and

ii) subjecting the antibody or fragment of step (i) to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody or fragment.

-   A39. A method for making an antibody, or an antigen-binding fragment    thereof, the method comprising steps of:

i) providing an antibody or a fragment that comprises a heavy chainvariable region having an amino acid sequence set forth in SEQ ID NO:106; and

ii) subjecting the antibody or fragment of step (i) to affinitymaturation and/or optimization, so as to provide an affinity maturedand/or optimized antibody or fragment.

-   A40. The method of any one of embodiments A34-A39, wherein step (ii)    comprises mutagenesis.-   A41. The method of embodiment A40, wherein the mutagenesis is within    a CDR.-   A42. The method of embodiment A40, wherein the mutagenesis is within    a variable region.-   A43. The method of embodiment A40, wherein the mutagenesis is within    a constant region.-   A44. The method of any one of embodiments A34-A43, wherein step (ii)    comprises light-chain shuffling.

1.-59. (canceled)
 60. A method of treating a subject suffering from orat risk of a condition, wherein the condition is a fibrotic condition, aliver disease, a kidney disease, a viral infection, a bacterialinfection, and/or a fungal infection, the method comprisingadministering to the subject an antibody, or an antigen-binding fragmentthereof, that specifically binds a human latent transforming growthfactor beta binding protein 1 (LTBP1)-proTGFβ complex and a human latenttransforming growth factor beta binding protein 3 (LTBP3)-proTGFβcomplex, comprising a heavy chain variable domain and a light chainvariable domain, wherein the heavy chain variable domain comprises: aheavy chain complementary determining region 1 (CDRH1) comprising anamino acid sequence set forth as SEQ ID NO: 166, a heavy chaincomplementary determining region 2 (CDRH2) comprising an amino acidsequence set forth as SEQ ID NO: 167, and a heavy chain complementarydetermining region 3 (CDRH3) comprising an amino acid sequence set forthas SEQ ID NO: 168; and wherein the light chain variable domaincomprises: a light chain complementary determining region 1 (CDRL1)comprising an amino acid sequence set forth as SEQ ID NO: 169, a lightchain complementary determining region 2 (CDRL2) comprising an aminoacid sequence set forth as SEQ ID NO: 170, and, a light chaincomplementary determining region 3 (CDRL3) comprising an amino acidsequence set forth as SEQ ID NO:
 171. 61. The method of claim 60,wherein the heavy chain variable domain comprises an amino acid sequenceset forth as SEQ ID NO: 318 and the light chain variable domaincomprises an amino acid sequence set forth as SEQ ID NO:
 319. 62. Themethod of claim 60, wherein the heavy chain variable domain consists ofan amino acid sequence set forth as SEQ ID NO: 318 and the light chainvariable domain consists of an amino acid sequence set forth as SEQ IDNO:
 319. 63. The method of claim 60, wherein the fibrotic condition isan organ fibrosis.
 64. The method of claim 63, wherein the organfibrosis is selected from the group consisting of kidney fibrosis, liverfibrosis, lung fibrosis, cardiac fibrosis, pancreatic fibrosis, skinfibrosis, scleroderma, muscle fibrosis, uterine fibroids, andendometriosis.
 65. The method of claim 60, wherein the fibroticcondition is a fibrotic disorder comprising chronic inflammation. 66.The method of claim 65, wherein the fibrotic disorder comprising chronicinflammation is a muscular dystrophy, multiple sclerosis, or cysticfibrosis.
 67. The method of claim 66, wherein the muscular dystrophy isDuechenne muscular dystrophy (DMD).
 68. The method of claim 60, whereinthe fibrotic condition is Alport syndrome.
 69. The method of claim 60,wherein the fibrotic condition is fibrosis, fibroids, desmoplasia, oramyotrophic lateral sclerosis (ALS).
 70. The method of claim 60, whereinthe condition is a liver disease.
 71. The method of claim 70, whereinthe liver disease is selected from the group consisting of non-alcoholicfatty liver disease (NAFLD), e.g., non-alcoholic fatty liver (NAFL) andnon-alcoholic steatohepatitis (NASH), which may include: noncirrhoticNASH with liver fibrosis, liver cirrhosis, NASH with compensatedcirrhosis, NASH with decompensated cirrhosis, liver inflammation withfibrosis, liver inflammation without fibrosis; stage 2 and 3 liverfibrosis, stage 4 fibrosis (NASH cirrhosis or cirrhotic NASH withfibrosis), primary biliary cholangitis (PBC) (formerly known as primarybiliary cirrhosis), and primary sclerosing cholangitis (PSC).
 72. Themethod of claim 70, further comprising administering administering amyostatin inhibitor to the subject.
 73. The method of claim 60, whereinthe condition is a kidney disease.
 74. The method of claim 73, whereinthe kidney disease is selected from the group consisting of IgAnephropathy, focal and segmental glomerulosclerosis, crescenticglomerulonephritis, lupus nephritis, and diabetic nephropathy.
 75. Themethod of claim 60, wherein the administering is subcutaneousadministration.
 76. A method of treating a subject suffering from or atrisk of a condition, wherein the condition is a fibrotic condition, aliver disease, a kidney disease, a viral infection, a bacterialinfection, and/or a fungal infection, the method comprisingadministering to the subject an antibody, or an antigen-binding fragmentthereof, that specifically binds a human latent transforming growthfactor beta binding protein 1 (LTBP1)-proTGFβ complex and a human latenttransforming growth factor beta binding protein 3 (LTBP3)-proTGFβcomplex, comprising a heavy chain variable domain and a light chainvariable domain, wherein the heavy chain variable domain comprises anamino acid sequence having at least 90% identity to SEQ ID NO: 318 andthe light chain variable domain comprises an amino acid sequence havingat least 90% identity to SEQ ID NO:
 319. 77. The method of claim 76,wherein the heavy chain variable domain comprises: a heavy chaincomplementary determining region 1 (CDRH1) comprising an amino acidsequence set forth as SEQ ID NO: 166, a heavy chain complementarydetermining region 2 (CDRH2) comprising an amino acid sequence set forthas SEQ ID NO: 167, and a heavy chain complementary determining region 3(CDRH3) comprising an amino acid sequence set forth as SEQ ID NO: 168;and wherein the light chain variable domain comprises: a light chaincomplementary determining region 1 (CDRL1) comprising an amino acidsequence set forth as SEQ ID NO: 169, a light chain complementarydetermining region 2 (CDRL2) comprising an amino acid sequence set forthas SEQ ID NO: 170, and, a light chain complementary determining region 3(CDRL3) comprising an amino acid sequence set forth as SEQ ID NO: 171.78. The method of claim 76, wherein the heavy chain variable domaincomprises an amino acid sequence set forth as SEQ ID NO: 318 and thelight chain variable domain comprises an amino acid sequence set forthas SEQ ID NO:
 319. 79. The method of claim 76, wherein the fibroticcondition is an organ fibrosis.
 80. The method of claim 79, wherein theorgan fibrosis is selected from the group consisting of kidney fibrosis,liver fibrosis, lung fibrosis, cardiac fibrosis, pancreatic fibrosis,skin fibrosis, scleroderma, muscle fibrosis, uterine fibroids, andendometriosis.
 81. The method of claim 76, wherein the fibroticcondition is a fibrotic disorder comprising chronic inflammation. 82.The method of claim 81, wherein the fibrotic disorder comprising chronicinflammation is a muscular dystrophy, multiple sclerosis, or cysticfibrosis.
 83. The method of claim 82, wherein the muscular dystrophy isDuechenne muscular dystrophy (DMD).
 84. The method of claim 76, whereinthe fibrotic condition is Alport syndrome.
 85. The method of claim 76,wherein the fibrotic condition is fibrosis, fibroids, desmoplasia, oramyotrophic lateral sclerosis (ALS).
 86. The method of claim 76, whereinthe condition is a liver disease.
 87. The method of claim 86, whereinthe liver disease is selected from the group consisting of non-alcoholicfatty liver disease (NAFLD), e.g., non-alcoholic fatty liver (NAFL) andnon-alcoholic steatohepatitis (NASH), which may include: noncirrhoticNASH with liver fibrosis, liver cirrhosis, NASH with compensatedcirrhosis, NASH with decompensated cirrhosis, liver inflammation withfibrosis, liver inflammation without fibrosis; stage 2 and 3 liverfibrosis, stage 4 fibrosis (NASH cirrhosis or cirrhotic NASH withfibrosis), primary biliary cholangitis (PBC) (formerly known as primarybiliary cirrhosis), and primary sclerosing cholangitis (PSC).
 88. Themethod of claim 86, further comprising administering administering amyostatin inhibitor to the subject.
 89. The method of claim 76, whereinthe condition is a kidney disease.
 90. The method of claim 89, whereinthe kidney disease is selected from the group consisting of IgAnephropathy, focal and segmental glomerulosclerosis, crescenticglomerulonephritis, lupus nephritis, and diabetic nephropathy.
 91. Themethod of claim 76, wherein the administering is subcutaneousadministration.